Selection Guide.................................... ........................................ .................................... 408 Products............................................................................................................................ 413
AllFermentasproductsare manufacturedinclassDcleanroom facilities,qualiedandce facilities, qualiedandcertiedas rtiedas perEUdirectivesandISPEguid perEUdirective sandISPEguidelines. elines. Fermentasqualityassurancei Fermentasqual ityassuranceiscarried scarried outaccordingtoISO9001qualityand ISO14001environmentalmanagement systems,guaranteeingbatch-to-batch reproducibility.Integrationofourclean roomandISOsystemsensuresstability andtheabsenceofcontaminantsinall ofourproducts.
S I S E R O H P O R T C E L E A N D
NoLimits™ Individual DNA Fragments and Custom DNA Ladders ............................ 413 GeneRuler™ and O’GeneRuler™ DNA Ladders (10-20000bp) .................................... 414 GeneRuler™1kbDNALadder.................................................................................... 415 GeneRuler™1kbPlusDNALadder............................................................................. 1kbPlusDNALadder............................................................................. 415 GeneRuler™DNALadderMix..................................................................................... 415 GeneRuler™100bpDNALadder................................................................................ 415 GeneRuler™100bpPlusDNALadder........................................................................ 415 GeneRuler™50bpDNAL adder....................................... ....................................... ........................................ ... 415 415 GeneRuler™UltraLowRangeDNALadder.................................................................. 415 GeneRuler™LowRangeDNALadder.......................................................................... 415 GeneRuler™HighRangeDNALadder......................................................................... 415 GeneRuler™Expres sDNALad der................................... ................................... ......................................... ... 415 MassRuler™ DNA Ladders, ready‑to‑use(80 -10000bp -10000bp ).................................. .................................. ......... 418 MassRuler™LowRangeDN ALadder..................................... ALadder..................................... ..................................... 418 MassRuler™ExpressLRForwardDNALadder............................................................ 418 MassRuler™ExpressLRReverseDNALadder............................................................ 418 MassRuler™HighRangeDNALadder......................................................................... 418 MassRuler™ExpressH RForwardD NALadd er..................................... ..................................... ....................... 418 MassRuler™ExpressHRReverseDNALadder............................................................ 418 MassRuler™DNALadderMix..................................................................................... 418 MassRuler™ExpressForwardDNALadderMix........................................................... 418 MassRuler™ExpressReverseDNALadderMix........................................................... 418 FastRuler™ DNA Ladders, ready‑to‑use (10-10000bp (10-10000bp ).................................... .................................... ......... 420 FastRuler™UltraLowRa ngeDNAL adder............................................ ....................... 420 FastRuler™LowRangeD NALadd er.......................................................... ................. 420 FastRuler™MiddleRange DNALad der................................................ ....................... 420 FastRuler™HighRangeD NALadd er....................................... ....................................... .................................... 420 ZipRuler™ Express DNA Ladder Set, ready‑to‑use(100-20000bp )......................... 421 O’RangeRuler™ DNA Ladders, ready‑to‑use (10-6000bp) .......................................422 O’RangeRuler™5bpDNALa dder.................................... .................................... ........................................ ... 422 O’RangeRuler™10bpDNALadder.............................................................................422 O’RangeRuler™20bpDNAL adder......................................... ......................................... .................................... 422 O’RangeRuler™50bpDNAL adder......................................... ......................................... .................................... 422 O’RangeRuler™100bpDNALadd er....................................... ....................................... .................................... 422 O’RangeRuler™200bpDNALadder..........................................................................422 O’RangeRuler™500bpDNALadder..........................................................................422 O’RangeRuler™100+500bpD NALadde r.................................... .................................... .............................. 422 Conventional Lambda DNA Markers (15-48502bp )................................... ................................... ............... 424 LambdaDN A/ EcoRIMar ker,1......................................... ker,1......................................... ........................................ .. 424 LambdaDN A/ HindIIIMa rker,2........................................ rker,2........................................ ........................................ .. 424 LambdaDN A/ EcoRI +HindI IIMarker,3.................................... .................................... ................................... 424 Lambda–p UCMIxMarker,4................................................. ................................... 424 LambdaDN A/ Eco47I(Ava II)M arker,13........................................ ........................................ ............................. 424 LambdaDN A/ Eco91I(Bs tEII )Marker,15....................................... ....................................... ............................. 424 LambdaDNA/Eco130I(StyI)Marker,16 ........................................ ............................. 424 LambdaMixMa rker,19..................................... ..................................... ........................................ ................ 424 LambdaDN A/ PstIMarker,24................................... PstIMarker,24................................... ......................................... ........ 424 Conventional Phage and Plasmid DNA Markers (8-1353bp) ....................................426 pBR322DN A/ BsuRI( HaeIII )Marker,5.................................... .................................... ................................... 426 pUCMixMarke r,8...................................... ...................................... ......................................... ...................... 426 F X174DNA/BsuRI(HaeIII)Marker,9........................................................................426 X174DNA/Hin fIMarker,10................................... ................................... ......................................... ........ 426 F X174DNA/Hin pBR322DN A/A luIMarker,20.................................... .................................... ........................................ ........ 426 pUC19DNA/MspI(HpaII)Marker,23.........................................................................426 Loading Dyes ..................................... ......................................... .................................. 428 TopVision™ Agarose.....................................................................................................430 Electrophoresis Buffers..............................................................................................430 Protocols and Recommendations...............................................................................431 9.1.GeneralrecommendationsforDNAelectrophoresis....................................................431 9.2.Preparationo fagarosegelsfo rDNAelect rophoresis........................................ rophoresis........................................ .......... 431 9.3.PreparationofgelsforPAGE.....................................................................................432 9.4.PreparationofDNAsamplesforelectrophoresis........................................................433 9.5.PreparationofDNAladders/markersforelectrophoresis............................................433 9.6.LabelingofDNAladders/markers.............................................................................433 9.7.SeparationofExpressDNAladdersindifferentelectrophoresisconditions..................434 Troubleshooting Troubles hooting Guide...................................................................................................435
9
Selection Guide PProduct lines of DNA ladders and markers Features DNA ladder/marker group
Separation Range, bp time on agarose
™
NoLimits Custom 10-20000 DNA Ladders
GeneRuler™ and O’GeneRuler™ DNA Ladders
10-48502
S I S E R O H P O R T C E L E A N D . 9
9
FastRuler™ DNA Ladders
O’RangeRuler™ DNA Ladders
80-10000 80-10000
10-10000 10-10000
10-6000
Formulation
P AG E
Fast
Individual, Any chromatoInTEbufferor Fermentas graphy-puried ready-to-use LoadingDye DNAfragments uponrequest
Sizingofbroad rangeDNA fragments
Mixtureof 6XDNA individual, Loading InTEbufferor chromatoDyeor ready-to-use graphy-puried 6XOrange DNAfragments LoadingDye
10-45min 10-45min
AccurateDNA quantication
Mixtureof individual, 6X chromatoReady-to-use MassRuler™ graphy-puried LoadingDye DNAfragments
8-14min 8-14min
Fastseparation inshortrun
Mixtureof 6X 5individual, MassRuler™ chromatoReady-to-use LoadingDye, graphy-puried 6XOrange DNAfragments LoadingDye
45min1.5h
10min1.5h
ZipRuler Express 100-20000 100-20000 10-20min 10-20min DNA Ladders
Labeling* with
Page
Klenow
–
413
–
–
414
–
–
–
418
–
420
Ligation Stepladdersof productsof 5,10,20,50, 10,15,20, 6XOrange Ready-to-use 100,200,500bp 50,100,200, LoadingDye increments 500bpDNA fragments
–
–
–
–
422
Mixtureof Fastandaccurate individual, sizingofbroad 6XOrange chromatoReady-to-use rangeDNA LoadingDye graphy-puried fragments DNAfragments
–
–
–
421
InTEbufferor 6XDNA ready-to-use LoadingDye
–
–
424
DigestedPhage InTEbufferor 6XDNA Classicalmarkers andplasmid ready-to-use LoadingDye DNA
–
426
45min-18h n-18h Classical Classicalmarke markers rs
45min1.5h
Quanti‑ cation
T4 PNK
Formulated according Composition tocustomer dependent specications. Bulkorders
™
Conventional 15-48502 15-48502 Lambda DNA Markers Conventional Phage and Plasmid 8-1353 DNA Markers
Origin
Electrophoresis Agarose
™
MassRuler DNA Ladders
Main featur e
Applications Loading dyes supplied
Digested LambdaDNA
Note NotalltheDNAladders/markersofthegrouparesuitablefortheindicatedapplication. *OnlyDNAladders/markerssuppliedinTEbuffercanbelabeled.
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see p.557 Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS Range selection for DNA Ladders and Markers 10 bp
100 bp
1 kb
10 kb
50 kb GeneRuler™ Ultra Low Range DNA Ladder, #SM1211/2/3,p.417 O’GeneRuler™ Ultra Low Range DNA Ladder, #SM1223,p.417
10-300bp
GeneRuler™ Low Range DNA Ladder, #SM1191/2/3,p.417 O’GeneRuler™ Low Range DNA Ladder, #SM1203,p.417
25-700bp
GeneRuler™ 50 bp DNA Ladder, #SM0371/2/3,p.416
50-1000bp
O’GeneRuler™ 50 bp DNA Ladder, #SM1133,p.416 GeneRuler™ 1 kb Plus DNA Ladder, #SM1331/2/3/4,p.416
75-20000bp
O’GeneRuler™ 1 kb Plus DNA Ladder, #SM1343,p.416 GeneRuler™ 100 bp DNA Ladder, #SM0241/2/3/4,p.416
100-1000bp
O’GeneRuler™ 100 bp DNA Ladder, #SM1143,p.416 GeneRuler™ 100 bp Plus DNA Ladder, #SM0321/2/3/4,p.416
100-3000bp
O’GeneRuler™ 100 bp Plus DNA Ladder, #SM1153,p.416 GeneRuler™ Express DNA Ladder, #SM1551/2/3,p.417
100-5000bp
O’GeneRuler™ Express DNA Ladder, #SM1563,p.417 GeneRuler™ DNA Ladder Mix, #SM0331/2/3/4,p.416
100-10000bp
O’GeneRuler™ DNA Ladder Mix, #SM1173,p.416 GeneRuler™ 1 kb DNA Ladder, #SM0311/2/3/4,p.416
250-10000bp
O’GeneRuler™ 1kb DNA Ladder, #SM1163,p.416
10171-48502bp
MassRuler™ Low Range DNA Ladder, #SM0383,p.419
80-1031bp 100-1000bp 100-1000bp
MassRuler™ Express LR Forward DNA Ladder, #SM1263,p.419 MassRuler™ Express LR Reverse DNA Ladder, #SM1273,p.419 MassRuler™ DNA Ladder Mix, #SM0403,p.419 MassRuler™ Express Forward DNA Ladder Mix, #SM1283,p.419
80-10000bp 100-10000bp 100-10000bp
MassRuler™ Express Reverse DNA Ladder Mix, #SM1293,p.419 MassRuler™ High Range DNA Ladder, #SM0393,p.419 MassRuler™ Express HR Forward DNA Ladder, #SM1243,p.419
1500-10000bp 1500-10000bp 1500-10000bp
MassRuler™ Express HR Reverse DNA Ladder, #SM1253,p.419 FastRuler™ Ultra Low Range DNA Ladder, #SM1233,p.420
10-200bp
FastRuler™ Low Range DNA Ladder, #SM1103,p.420 FastRuler™ Middle Range DNA Ladder, #SM1113,p.420
50-1500bp 100-5000bp
FastRuler™ High Range DNA Ladder, #SM1123,p.420
500-10000bp
O’RangeRuler™ 5 bp DNA Ladder, #SM1303,p.423
10-100bp 10-150bp 20-300bp 50-1000bp 100-1500bp 100-6000bp
O’RangeRuler™ 10 bp DNA Ladder, #SM1313,p.423 O’RangeRuler™ 20 bp DNA Ladder, #SM1323,p.423 O’RangeRuler™ 50 bp DNA Ladder, #SM0613,p.423 O’RangeRuler™ 100 bp DNA Ladder, #SM0623,p.423 O’RangeRuler™ 100+500 bp DNA Ladder, #SM0653,p.423
9
O’RangeRuler™ 200 bp DNA Ladder, #SM0633,p.423
200-3000bp
O’RangeRuler™ 500bp DNA Ladder, #SM0643,p.423
500-6000bp
ZipRuler™ Express DNA Ladder 1, #SM1373,p.421
100-10000bp 200-20000bp
ZipRuler™ Express DNA Ladder 2, #SM1373,p.421 pBR322 DNA/BsuRI Marker, 5, #SM0271,p.426 pBR322 DNA/AluI Marke r, 20, #SM0121/3,p.427
8-587bp 11-908bp 15-11501bp 19-1118bp 23-8126bp 24-726bp 26-501bp
Lambda DNA/PstI Marke r, 24,#SM0361,p.425 pUC Mix Marker, 8, #SM0301/2/3,p.427 Lambda DNA/Eco47I Mar ker, 13, #SM1051,p.425 FX174 DNA/HinfI Marke r, 10, #SM0261,p.427 pUC19 DNA/MspI Marker, 23, #SM0221/2/3,p.427 FX174 DNA/BsuRI Marke r, 9, #SM0251/2/3,p.427 Lambda – pUC Mix Marker, 4, #SM0291,p.425 Lambda DNA/Eco130I Marke r, 16,#SM0161,p.425 Lambda DNA/Eco91I Marke r, 15, #SM0111,p.425 Lambda DNA/HindIII Mark er, 2,#SM0101/2/3,p.425 Lambda DNA/EcoRI+HindIII Marker, 3, #SM0191/2/3,p.425 Lambda Mix Marker, 19, #SM0231,p.425 Lambda DNA/EcoRI Marke r, 1, #SM0281,p.425
72-1353bp 74-19329bp 74-19329bp 117-8453bp 125-23130bp 125-21226bp 1503-48502bp 3530-21226bp
10bp
GeneRuler™ High Range DNA Ladder, #SM1351/2/3,p.417
100bp
1kb
10kb
50kb
Bulkquantitiesandcustomformulationsavailableuponrequest
409
Reference for DNA Ladders and Markers
50 kb
S I S E R O H P O R T C E L E A N D . 9
9
7 1 e . g e 4 n 7 g p , a 1 n 3 R 4 . a / 2 w p R / , o 1 3 w L 1 2 o 2 L 2 1 a r 1 a M t l M r U t S l # S U ™ #
r r ™ r e e l e r d d e l d u d u a R e a R L L n e e A A n e N G ’ N G D O D
r e r e d d d a d L a L A A N N D D e e g g n n a a 7 R R 1 w 4 . o 7 w p L 1 o , ™ 4 L 3 r . ™ / e p 2 r / l , u 3 e l 1 R 0 u 9 1 e 2 R n 1 e 1 e n G e M ’ M S G S # O #
r e r e d d d a d L a L A A N N D D p p 6 b b 1 0 4 0 . 0 6 0 p 1 ™ 1 1 3 r . ™ / e 4 2 p r / l u 3 e l 1 R 4 u 4 2 e 1 R 0 n e e 1 n M G e S ’ M G # O S #
r r e e d d d d a L a L A A N N D D s u s l u P l P p b p 6 b 1 0 4 . 0 0 1 6 p 0 , 1 3 r ™ 1 4 . ™ / e p 2 r / l e u 3 l 1 R 5 u 2 3 e 1 R 0 n e e 1 n M M G e ’ S G S # O #
r e r e d d d d a L a L A A N N D D s s s 7 e s r e p r 1 4 x . p E 7 p x , 1 E 3 ™ 4 r . ™ / e p 2 r / l e u 3 l 1 R 6 u 5 5 e 5 R 1 n e e 1 n M G e ’ M S G S # O #
x i x M i M r e r e d d d a d L a L 6 1 A A 4 . N D 6 N p , ™ 1 D 3 4 r . ™ / e p 2 r / l , u e 3 l 1 R 7 u 3 3 e 1 R n 0 e 1 e n G e M ’ M S G S # O #
™ r e r e l d u R d e a L n e G A ’ N O D 6 r b 1 . e 6 k 4 p , d 1 3 d 1 4 . ™ / a p 2 r , / L e l 1 A 3 u 1 N 6 3 D 1 R 0 b 1 e n e M k M S G S # 1 #
e g n a 7 R 1 h 4 . g p i , H 3 ™ r e / 2 r / e 1 l d 5 u d a 3 R L e A 1 n M e N G D S # 50 kb
10 kb
10 kb
1 kb
1 kb
100 bp
100 bp
10 bp
50 kb
e g n a R w 9 o L 1 4 . ™ r p e , r e d l d 3 u a 8 R L 3 s 0 s a A N M S M D #
d e r a s r e 9 w 9 r 1 v e 1 o 4 F p . R 4 . p R , R , L 3 L 3 6 s 7 s 2 s 2 s e e 1 r 1 M r M p p x S x S # E E # ™ r ™ r e r e r e e l d l d u d u d a a R L R L s s s s A a N a A N M D M D
A N 9 D 1 4 . ™ x p r i , e l M 3 u r 0 R e 4 s d 0 s M a d S a M L #
9 9 1 1 4 . 4 . d p e p r , s , a 3 r 3 e w 8 v 9 r 2 e 2 o F 1 R 1 M s M s S s S s e r # e r #
p x p x i i x E M x E M r ™ ™ r e r e r e e l d l d d u a u d a R L R L s s s s A a N a A N D M D M
e g n a R h g 9 i H 1 4 . ™ r p e , r e d l d 3 u a 9 R L 3 s 0 s M a A N S M D #
d e r s a r w 9 e 9 v r 1 e 1 o F 4 R 4 . . p R p , R , H 3 H 3 4 s 5 s 2 s 2 s e e 1 r 1 M r M p p x S x S # E E # ™ r ™ r e r e r e e l d l d u d u d a a R L R L s s s s A a N a A N M D M D
10 bp
e g n a R w o L a r 0 t l 2 U r 4 . ™ e p , r d 3 e d l a 3 u L 2 R 1 t A M s N S a F D #
e g n a R 0 w 2 o L r 4 . ™ e p , r d 3 e d l a 0 u L 1 R 1 t A M s N S a F D #
e g n a R e l d 0 d 2 i M r 4 . ™ e p , r d 3 e d l a 1 u L 1 R 1 t A M s N S a F D #
e g n a R h 0 g 2 i H r 4 . ™ e p , r d 3 e d l a 2 u L 1 R 1 t A M s N S a F D #
50 kb
10 kb
10 kb
1 kb
1 kb
100 bp
100 bp
10 bp
10 bp
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410
r e r e d d d d a L a L A A N N D D 6 p b p 1 b 4 . 0 5 p 0 , ™ 6 5 3 1 r ™ / e 4 . 2 r / l p u e , l 1 7 R 3 u 3 e 3 R 0 n 1 e e n G e M ’ M S G S # O #
r e r d e d d d a L a L A A N N D D s u s l u 6 P l P 1 b k 4 b . 1 6 k p , ™ 1 1 3 r 4 . ™ / l p 2 e r , e / 1 u 3 l R 4 u 3 e 3 3 R e 1 n e 1 n e M ’ M S G G # O S #
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
50 kb
p b 5 3 ™ 2 r e . l r 4 u e p , d 3 R e d 0 g a n L 3 a A 1 R N M ’ O D S #
p b 0 1
3
™ 2 r e . l r 4 u e p , d 3 R e d 1 g a n L 3 a A 1 R N M ’ O D S #
p b 0 2
3
™ 2 r e . l r 4 u e p , d 3 R e d 2 g a n L 3 a A 1 R N M ’ O D S #
p b 0 5
3
™ 2 r e . l r 4 u e p , d 3 R e d 1 g a n L 6 a A 0 R N M ’ O D S #
p b 0 0 1 3 ™ 2 r e . l r 4 u e p , d 3 R e d 2 g a n L 6 a A 0 R N M ’ O D S #
p b 0 0 5 + 0 0 1 3 ™ 2 r e . l r 4 u e p , d 3 R e d 5 g a n L 6 a A 0 R N M ’ O D S #
p b 0 0 2 3 ™ 2 r e . l r 4 u e p , d 3 R e d 3 g a n L 6 a A 0 R N M ’ O D S #
p b 0 0 5 3 ™ 2 r e . l r 4 u e p , d 3 R e d 4 g a n L 6 a A 0 R N M ’ O D S #
A N D s s e r 1 2 p 4 x . E p , ™ 3 r 1 7 e r 3 l e u d 1 R d M p a S i Z L #
A N D s s e r 1 2 p 4 x . E p , ™ 3 r 2 7 e r 3 l e u d 1 R d M p a S i Z L
50 kb
#
10 kb
10 kb
1 kb
1 kb
100 bp
100 bp
10 bp
10 bp
50 kb
5 , I R u s 6 B 2 / A 4 . N p , D 1 7 2 2 2 3 0 R M B S p #
0 2 , I 7 u l 2 A . / 4 A p , N 3 D / 1 2 2 1 2 3 0 R M B S p #
4 2 , I t s 5 P 2 / A 4 . N p , D 1 a 6 d 3 b 0 M m a L S #
7 2 4 . p , 3 / 2 8 / , 1 x 0 i 3 M 0 C M U S p #
3 1 , I 7 4 o c 5 E 2 / A 4 . N p , D 1 a 5 d 0 b 1 M m a L S #
0 1 , I f n 7 i H 2 / 4 . A p N 1 , D 6 4 2 7 1 0 X M S F #
3 2 7 , 2 I 4 p . s p , M 3 / / A 2 N / D 1 2 9 2 1 0 C M U S p #
9 , 7 I R 2 4 u . s p , B / / 3 A 2 N / D 1 5 4 2 7 1 0 X M S
50 kb
F #
10 kb
10 kb
1 kb
1 kb
100 bp
100 bp
10 bp
9
* Indicatesthefragmentswithcohesiveendsofthe12ntcossitesofλDNAthatmayannealandformanadditionalband. Thesefragments(showninitalic )canbeseparatedbyheatingat65°Cfor5minandthencoolingonicefor3min. Indicatesapossibleadditionalbandduetoannealingofcohesiveendsofthefragmentsfrom12ntcossitesof λDNA. Referencebandsappearinred.
10 bp
Bulkquantitiesandcustomformulationsavailableuponrequest
411
4 , x i M 5 C 2 U 4 . p p , – 1 a 9 d 2 b 0 m M a L S #
6 1 , I 0 3 1 o c 5 E 2 / A 4 . N p , D 1 a 6 d 1 b 0 m M a L S #
5 1 , I 1 9 o c 5 E 2 / A 4 . N p , D 1 a 1 d 1 b 0 m M a L S #
2 , I I I 5 d 2 n 4 i . H p / , A 3 N / 2 D / a 1 d 0 b 1 0 m M a L S #
3 , I I I d n i H + I 5 R 2 o c 4 . E p / , A 3 N / 2 D / a 1 d 9 b 1 0 m M a L S #
9 5 1 , 2 4 . x i p , M 1 a 3 d 2 b 0 m M a L S #
1 , I R o c 5 E 2 / A 4 . N p , D 1 a 8 d 2 b 0 m M a L S #
50 kb 50 kb
10 kb 10 kb
S I S E R O H P O R T C E L E A N D . 9
1 kb 1 kb
100 bp 100 bp
10 bp 10 bp
* Indicatesthefragmentswithcohesiveendsofthe12ntcossitesofλDNAthatmayannealandformanadditionalband.
9
Thesefragments(showninitalic )canbeseparatedbyheatingat65°Cfor5minandthencoolingonicefor3min. Indicatesapossibleadditionalbandduetoannealingofcohesiveendsofthefragmentsfrom12ntcossitesofλDNA. Referencebandsappearinred.
www.fermentas.com
412
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
Products NoLimits™ Individual DNA Fragments and Custom DNA Ladders Collection of Individual DNA Fragments
DNA fragment size 10 bp 15 bp 20 bp 25 bp 35 bp 50 bp 75 bp 100 bp 150 bp 200 bp 250 bp 300 bp 400 bp 500 bp 600 bp 700 bp
Cat. #
Amount, µg
SM1391 SM1381 SM1401 SM1761 SM1411 SM1421 SM1431 SM1441 SM1601 SM1611 SM1451 SM1621 SM1631 SM1641 SM1461 SM1651
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Description NoLimits™isacollectionof36highly-puried individualDNAfragmentsrangingfrom 10to20,000bp. NoLimits™DNAfragmentsareproducedusing specicallydesignedplasmidspuriedbya patentedtechnology.PlasmidDNAisdigested withPureExtreme®restrictionenzymesandthe individualDNAfragmentsarechromatographypuriedfromthedigestionmixture.Asaresult, weofferexceptionallypureDNAfragments thatarefreeofanytruncatedordegraded molecules.OurcustomDNAfragmentsare thebestchoiceforapplicationsrangingfrom electrophoresis(bothgelandcapillary)toHPLC andbeyond.
750 bp
SM1471
10
800 bp
SM1481
10
850 bp
SM1661
10
900 bp
SM1491
10
1000 bp
SM1671
10
1200 bp
SM1681
10
1500 bp
SM1691
10
Features • Chromatography-puriedindividualDNA fragments. • Broadrange–10-20000bp. • Sharppeaksduringcapillaryelectrophoresis, sharpbandsaftergelelectrophoresis. • PreciseDNAconcentration.
2000 bp
SM1701
10
2500 bp
SM1571
10
3000 bp
SM1711
10
3500 bp
SM1501
10
4000 bp
SM1721
10
5000 bp
SM1731
10
6000 bp
SM1511
10
7000 bp
SM1741
10
8000 bp
SM1521
10
10000 bp
SM1751
10
15000 bp
SM1531
10
17000 bp
SM1771
10
20000 bp
SM1541
10
Applications • Gelelectrophoresis. • Capillaryelectrophoresis. • DNAquantication. • HPLCanalysis. • ApplicationswhereacustomDNAsize standardisrequired. Concentration 0.5µg/µl Storage Buffer(TEbuffer) 10mMTris-HCl(pH7.6)with1mMEDTA. Quality Control TestedonanAgilent2100bioanalyzerandby appropriategelelectrophoresis.DNAconcentration determinedspectrophotometrically.Theabsenceof nucleasesconrmedbyappropriatetests. Storage Storeat-20°C.
9
1.1%TopVision ™Agarose(#R0491),1XTAE,7V/cm,30min. NoLimits™DNAfragmentsfrom300bpto10kb.
NoLimits™ Custom DNA Ladders Service Bulkorderscanbecustomizedtotyourneeds: • IndividualDNAfragmentsormixtures. • Customladdersofanycombinationand concentration. • Referencebandsinacustom-madeDNAladder. • Flexibleformulations. • Ready-to-useversionspremixedwithany loadingdye( see p.428). • Frommicrogramtogramquantities.
How to Order? ContactyourlocalFermentasrepresentative: see Contactsonwww.fermentas.com
Bulkquantitiesandcustomformulationsavailableuponrequest
413
GeneRuler™ and O’GeneRuler™ DNA Ladders (10-20000bp) Related Products • • • • • • • •
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer T4 Polynucleotide Kinase ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free
p.484 p.428 p.430 p.430 p.244 p.469 p.477 p.476
S I S E R O H P O R T C E L E A N D . 9
9
Description GeneRuler™DNAladdersaremixturesofchromatography-puriedindividualDNAfragments. TheGeneRuler™lineincludesthemostpopular ladderssuchasthe100bpand1kbDNA Ladder. GeneRuler™DNAladdersareavailablein twoformats:conventional(TEbuffer)anda ready-to-useformat(premixedwith6XDNA LoadingDyewhichcontainsbromophenolblue andxylenecyanolFF). ConventionalversionscanbelabeledradioactivelywithT4PolynucleotideKinase(#EK0031). O’GeneRuler™DNAladdersareanother ready-to-useversionofGeneRuler™DNAladders.Theyarepremixedwith6XOrangeDNA LoadingDye,whichcontainsxylenecyanolFF andorangeG.Ina1%agarosegelorangeG dyemigratesat50bp.ThereforeO’GeneRuler ™ DNAladdersareidealwhenvisualizationof smallDNAfragmentsisimportant. GeneRuler™ Ultra Low Range and Low Range DNA laddersareidealfordenaturing polyacrylamidegelelectrophoresis(Fig.9.4). GeneRuler™ and O’GeneRuler™ Ultra Low Range DNA laddersareidealforanalysisof siRNA. GeneRuler™ High Range DNA Ladderis designedforfastsizingofhighmolecularweight DNAfragments(1.5hin0.4%agarosegel). GeneRuler™ and O’GeneRuler™ Express DNA laddersaredesignedforfastseparation (in5-15minat23V/cm)underawiderangeof electrophoresisconditions,indifferentbuffers, voltagesorgelpercentages. Theladdersaresupplied(dependingontheversion)eitherwith6XDNALoadingDyeorwith6X OrangeDNALoadingDyeforsampleDNA.
Features • IdealforbothDNAsizingandapproximate quantication. • Sharpbands. • Assembledofchromatographypuried individualDNAfragments. • Brightreferencebands. • Ready-to-useladderscanbedirectlyloaded andarestableatroomtemperaturefor 6months. • SuppliedwithloadingdyeforsampleDNA. Storage Buffer(TEbuffer) 10mMTris-HCl(pH7.6)and1mMEDTA. GeneRuler™ Storage and Loading Buffer (for ready‑to‑use ladders) 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,0.005%xylene cyanolFFand10%glycerol. 6X DNA Loading Dye 10mMTris-HCl(pH7.6),0.03%bromophenol blue,0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. O’GeneRuler™ Storage and Loading Buffer 10mMTris-HCl(pH7.6),10mMEDTA, 0.025%orangeG,0.005%xylenecyanolFF and10%glycerol. 6X Orange DNA Loading Dye 10mMTris-HCl(pH7.6),0.15%orangeG, 0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.ConcentrationofeachDNAfragment andofthecompleteladderdeterminedspectrophotometrically.Theabsenceofnucleases conrmedbyappropriatetests. Storage Storeat-20°C. Ready-to-useversionscanbestoredatroom temperatureorat4°Cfor6months.
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.3. Preparation of gels for PAGE » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis » 9.7.SeparationofExpressDNAladdersin differentelectrophoresisconditions » 7.1. DNA/RNA end labeling
www.fermentas.com
414
p.431 p.431 p.432 p.433 p.433 p.434 p.380
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
DNA ladder
Cat. #
GeneRuler™ 1 kb DNA Ladder GeneRuler™ 1 kb DNA L adder, ready‑to‑use O’GeneRuler™ 1 kb DNA Ladder, ready‑to‑use GeneRuler™ 1 kb Plus DNA Ladder
SM0311 SM0312 SM0313 SM0314
SM1331 SM1332 SM1333 SM1334
Amount, µg
Applications, Loading, 0.5 µg/lane µg(µl)/lane
250(5x50) 1250(25x50) 250(5x50) 50
500 2500 500 100
250 (5x50)
500
250(5x50) 1250(25x50) 250(5x50) 50
500 2500 500 100
250 (5x50)
500
250(5x50) 1250(25x50) 250(5x50) 50
500 2500 500 100
250 (5x50)
500
50 250(5x50) 50 250(5x50
100 500 100 500
50
100
50 250(5x50) 50 250(5x50)
100 500 100 500
50
100
0.5
50 250(5x50)
100 500
0.5(1)
0.1
50
100
0.5 (5)
0.5
50 250(5x50)
50-100 250-500
0.5-1(1-2)
0.1
50
100
0.5-1 (5-10)
0.5
50 250(5x50)
50-100 250-500
0.5-1(1-2)
0.1
50
100
0.5-1 (5-10)
0.5
50 250(5x50)
100 500
0.5(1)
0.1
50
100
0.5 (5)
0.5
50 250(5x50)
100 500
0.5(1)
0.5
0.1
SM1163
GeneRuler™ 1 kb Plus DNA Ladder, ready‑to‑use O’GeneRuler™ 1 kb Plus DNA Ladder, SM1343 ready‑to‑use SM0331 GeneRuler™ DNA Ladder Mix SM0332 ™ GeneRuler DNA Ladder Mix, SM0333 ready‑to‑use SM0334 O’GeneRuler™ DNA Ladder Mix, SM1173 ready‑to‑use SM0241 GeneRuler™ 100 bp DNA Ladder SM0242 GeneRuler™ 100 bp DNA Ladder, SM0243 ready‑to‑use SM0244 O’GeneRuler™ 100 bp DNA Ladder, SM1143 ready‑to‑use SM0321 GeneRuler™ 100 bp Plus DNA Ladder SM0322 ™ GeneRuler 100 bp Plus DNA Ladder, SM0323 ready‑to‑use SM0324 O’GeneRuler™ 100 bp Plus DNA SM1153 Ladder, ready‑to‑use SM0371 GeneRuler™ 50 bp DNA Ladder SM0372 GeneRuler™ 50 bp DNA Ladder, SM0373 ready‑to‑use O’GeneRuler™ 50 bp DNA Ladder, SM1133 ready‑to‑use GeneRuler™ Ultra Low Range DNA SM1211 Ladder SM1212 ™ GeneRuler Ultra Low Range DNA SM1213 Ladder, ready‑to‑use O’GeneRuler™ Ultra Low Range DNA SM1223 Ladder, ready‑to‑use SM1191 GeneRuler™ Low Range DNA Ladder SM1192 GeneRuler™ Low Range DNA Ladder, SM1193 ready‑to‑use ™ O’GeneRuler Low Range DNA SM1203 Ladder, ready‑to‑use SM1351 GeneRuler™ High Range DNA Ladder SM1352 ™ GeneRuler High Range DNA Ladder, SM1353 ready‑to‑use SM1551 GeneRuler™ Express DNA Ladder SM1552 GeneRuler™ Express DNA Ladder, SM1553 ready‑to‑use O’GeneRuler™ Express DNA Ladder, SM1563 ready‑to‑use Suppliedwith: 6X DNA Loading Dye 6XOrangeDNALoadingDye
Concentration, µg/µl
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.1
50
100
Range, bp
Number of Agarose, fragments %
PAGE, %
0.5(1) 250-10000
14
0.7-1.2
–
75-20000
15
0.7-1.2
–
100-10000
21
0.7-1.2
–
100-1000
10
1.7-2.5
4-8
100-3000
14
1.7-2.5
–
50-1000
13
1.7-2.5
4-8
0.5(5)
0.5(1)
0.5(5)
0.5(1)
0.5(5)
0.5(1)
0.5(5)
0.5(1)
0.5(5)
9 10-300
11
4.5-5.0
8-10
25-700
10
2.5-3.0
8-10
1017148502
8
0.3-0.5
–
100-5000
9
1.7-2.5
–
0.5 (5)
1 ml/2 ml/10 ml 1ml/2ml/10ml
Bulkquantitiesandcustomformulationsavailableuponrequest
415
GeneRuler™ 1 kb DNA Ladder
GeneRuler™ 1 kb Plus DNA Ladder
GeneRuler™ DNA Ladder Mix
#SM0311/2/3
#SM1331/2/3
#SM0331/2/3 ™
O’GeneRuler 1 kb DNA Ladder, ready‑to‑use
O’GeneRuler 1 kb Plus DNA Ladder, ready‑to‑use
O’GeneRuler™ DNA Ladder Mix, ready‑to‑use
#SM1163
#SM1343
#SM1173
™
bp ng/0.5 µg
S I S E R O H P O R T C E L E A N D . 9
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
%
1000 0 8000 6000 5000 4000 3500 3000 2500 2000 1500
3 0.0 6.0 30.0 6.0 70.0 14.0 30.0 6.0 30.0 6.0 30.0 6.0 70.0 14.0 25.0 5.0 25.0 5.0 25.0 5.0
1000 750
60.0 25.0
12.0 5.0
500
25.0
5.0
250
25.0
5.0
bp ng/0.5 µg
20 000 10 000 7000 5000 4000 3000 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
4.0 4.0 4.0 15.0 4.0 4.0
2000
20.0
4.0
1500
80.0
16.0
1000
25.0
5.0
700 500 400 300 200 75
25.0 75.0 25.0 25.0 25.0 25.0
5.0 15.0 5.0 5.0 5.0 5.0
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
10 000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1200 1000 900 800 700 600 500 400 300 200 100
18.0 18.0 18.0 18.0 18.0 18.0 60.0 16.0 16.0 16.0 16.0 60.0 17.0 17.0 17.0 17.0 60.0 20.0 20.0 20.0 20.0
% 3 .6 3.6 3.6 3.6 3.6 3.6 12.0 3.2 3.2 3.2 3.2 12.0 3.4 3.4 3.4 3.4 12.0 4.0 4.0 4.0 4.0
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
GeneRuler™ 100 bp DNA Ladder
GeneRuler™ 100 bp Plus DNA Ladder
GeneRuler™ 50bp DNA Ladder
#SM0241/2/3
#SM0321/2/3
#SM0371/2/3 ™
O’GeneRuler™ 100 bp DNA Ladder, ready‑to‑use
O’GeneRuler 100 bp Plus DNA Ladder, ready‑to‑use
O’GeneRuler™ 50bp DNA Ladder, ready‑to‑use
#SM1143
#SM1153
#SM1133
bp ng/0.5 µg
9 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
1000 45.0 900 45.0 800 45.0 700 45.0 600 45.0 500 115.0 400 40.0 300 40.0 200 100
40.0 40.0
%
bp
9.0 9.0 9.0 9.0 9.0 23.0 8.0 8.0
1000 900 800 700 600 500 400
8.0 8.0
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1h
300
200 e d i m a l y r c a y l o p % 5
www.fermentas.com
bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
100
%
3000 2000
28.0 28.0
5.6 5.6
1500 1200 1000 900 800 700 600 500 400
28.0 28.0 80.0 27.0 27.0 27.0 27.0 80.0 30.0
5.6 5.6 16.0 5.4 5.4 5.4 5.4 16.0 6.0
300
30.0
6.0
200
30.0
6.0
100
30.0
6.0
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1h
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
416
20.0 20.0 20.0 75.0 20.0 20.0
bp ng/0.5 µg
%
bp
bp ng/0.5µg
3000 2000 1500 1200 1000 900 800 700 600 500 400 300
200 e d i m a l y r c a y l o p % 5
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 2
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
bp
1000 900 800 700 600 500 400
30.0 30.0 30.0 30.0 30.0 75.0 30.0
6.0 6.0 6.0 6.0 6.0 15.0 6.0
300 250 200 150
30.0 75.0 35.0 35.0
6.0 15.0 7.0 7.0
400
100
35.0
7.0
200
50
35.0
7.0
150
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1h
100
%
1000 900 800 700 600 500
300 250
100
e d i m a l y r c a y l o p % 5
50
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
GeneRuler™ Ultra Low Range DNA Ladder
GeneRuler™ Low Range DNA Ladder
GeneRuler™ High Range DNA Ladder
#SM1211/2/3
#SM1191/2/3
#SM1351/2/3
™
™
O’GeneRuler Ultra Low Range DNA Ladder, ready‑to‑use
O’GeneRuler Low Range DNA Ladder, ready‑to‑use
#SM1223
#SM1203 bp ng/0.5 µg 300 200 150
30.0 32.5 35.0
6.0 6.5 7.0
100 75
37.5 37.5
7.5 7.5
105.0
21.0
50 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5
bp
%
35 25 20 15
37.5 37.5 40.0 47.5
7.5 7.5 8.0 9.5
10
60.0
12.0
300 200 150 100 75 50 35 25 20 15
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1h
bp ng/0.5 µg
e d i m a l y r c a y l o p % 0 1
10
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 3
%
700 500 400
30.0 32.5 35.0
6.0 6.5 7.0
300
90.0
18.0
200
35.0
7.0
150
35.0
7.0
100 75
90.0 37.5
18.0 7.5
50
40.0
8.0
25
75.0
15.0
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1h
0.5µg/lane,20cmlengthgel, 1XTBE,8V/cm,3h
bp ng/0.5 µg
bp
%
700 500 400 300 200 150 100 75 50
25
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 4 . 0
48502 24508 20555 17000
80.0 88.0 80.0 59.0
16.0 17.7 15.9 11.8
15258 59.0 13825 50.0 12119 47.0
11.8 10.0 9.4
10 171 37.0
7.3
0.4µg/lane,8cmlengthgel, 1XTAE,3V/cm,1.5h
e d i m a l y r c a y l o p % 0 1
0.5µg/lane,20cmlengthgel, 1XTBE,8V/cm,3h
GeneRuler™ Express DNA Ladder #SM1551/2/3
O’GeneRuler™ Express DNA Ladder, ready‑to‑use
9
#SM1563 bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
%
5000
40.0
3000
40.0
8.0
2000 40.0 1500 100.0
8.0 20.0
1000 750
8.0
40.0 40.0
8.0 8.0
500
100.0
20.0
300
50.0
10.0
100
50.0
10.0
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,40min
5min 10min 15min
Figure 9.1. Fast separation of GeneRuler™ Express DNA Ladder at high voltage (23 V/cm). Electrophoresisconditions:0.5µg/lane,1%TopVision ™ Agarose(#R0491),1XTAE,23V/cm,5-15min.
Bulkquantitiesandcustomformulationsavailableuponrequest
417
MassRuler™ DNA Ladders, ready‑to‑use (80-10000bp) DNA ladder, ready‑to‑use
Cat. #
MassRuler™ Low Range DNA Ladder MassRuler™ Express LR Forward DNA Ladder MassRuler™ Express LR Reverse DNA Ladder MassRuler™ High Range DNA Ladder MassRuler™ Express HR Forward DNA Ladder MassRuler™ Express HR Reverse DNA Ladder MassRuler™ DNA Ladder Mix MassRuler™ Express Forward DNA Ladder Mix MassRuler™ Express Reverse DNA Ladder Mix Suppliedwith: 6XMassRuler™ DNA Loading Dye
• • • • •
SM0383 SM1263 SM1273 SM0393 SM1243 SM1253 SM0403 SM1283 SM1293
Volume, µl
Applications
Loading, µl/lane
60.8 28
2x500
50-200
5-20
2x500
50-200
5-20
2x500
50-200
5-20
Range, bp
Number of Agarose, fragments %
80-1031
11
1.7-2.5
100-1000
6
0.7-2.0
9
0.7-1.2
6
0.7-1.5
80-10000
20
1.0-1.2
100-10000
12
0.7-1.5
42.2 28.5 103 56.5
1500-10000
1 ml
Related Products
S I S E R O H P O R T C E L E A N D . 9
Concentration, ng/µl
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer Water, nuclease-free
p.484 p.428 p.430 p.430 p.476
Description MassRuler™DNAladdersarespeciallydesigned foraccuratequanticationandsizingofDNA fragmentsbyagarosegelelectrophoresis.The intensityofthefragmentineachladderiscalibratedagainstastandardthatguaranteesthe precisequantityofeachband.Theladdersare mixturesofchromatography-puried,individual DNAfragments. MassRuler™ExpressForwardandReverseDNA LaddersallowfastandreliableDNAquanticationinshortseparationdistancesundervarious electrophoresisconditions.Intheforward versions,themassofeachDNAfragment isdirectlyproportionaltothefragmentsize, whereasinthereverseversions,themassis inverselyproportionaltothefragment’ssize.
9
Features • AccurateDNAsizingandquantication. • Sharpbands. • Assembledofchromatographypuried individualDNAfragments. • Easy-to-rememberfragmentsizesand quantities. • Ready-to-use–premixedwithLoadingDye. • SuppliedwithloadingdyeforsampleDNA. Storage and Loading Buffer 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,10%glycerol. 6X MassRuler™ DNA Loading Dye 10mMTris-HCl(pH7.6),0.03%bromophenol blue,60%glyceroland60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.ConcentrationofeachDNAfragment andofthecompleteladderdeterminedspectrophotometrically.Theabsenceofnucleases conrmedbyappropriatetests. Storage Storeat-20°C(orat4°Corroomtemperature for6months).
A. MassRuler™ Express LR Forward DNA Ladder
[FU] 5000
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophortesis p.431 » 9.1.1.Recommendationsforaccuratein-gel quantication p.431 » 9.2.PreparationofagarosegelsforDNA electrophoresis p.431 » 9.4.PreparationofDNAsamplesfor electrophoresis p.433 » 9.7.SeparationofExpressDNAladders indifferentelectrophoresisconditions p.434
www.fermentas.com
418
B. MassRuler™ Express LR Reverse DNA Ladder [FU] 5000
4000
4000
3000
3000
2000
2000
1000
1000
0
0 40
60
80
100
40
60
80
100
Figure 9.2. Analysis of the MassRuler™ Express LR Forward and Reverse DNA Ladders (#SM1263 and #SM1273) using the Agilent 2100 Bioanalyzer DNA 1000 LabChip® Kit. A–analysisofMassRuler™ExpressLRForwardDNALadder B–analysisofMassRuler™ExpressLRReverseDNALadder
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
MassRuler™ Low Range DNA Ladder, ready‑to‑use
MassRuler™ Express LR Forward DNA Ladder, ready‑to‑use
#SM0383
MassRuler™ Express LR Reverse DNA Ladder, ready‑to‑use
#SM1263 #SM1273
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
ng/20 µl ng/15 µl ng/10 µl ng/5 µl b p
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
Forward
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
1031 900 800 700 600 500
200 180 160 140 120 200
150 135 120 105 90 150
100 90 80 70 60 100
50 45 40 35 30 50
400
80
60
40
20
300
60
45
30
15
Reverse
200
150
100
50
1000
1000
20
15
10
5
105 75
70 50
35 25
700 500
700 500
40 60
30 45
20 30
10 15
45 30 15
30 20 10
15 10 5
300 200 100
300 200 100
100 140 200
75 105 150
50 70 100
25 35 50
200
40
30
20
10
140 100
100 80
20 16
15 2
10 8
5 4
60 40 20
1%TopVision ™Agarose(#R0491) 10µl/lane,8cmlengthgel,1XTAE,7V/cm,30min
10µl/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
MassRuler™ High Range DNA Ladder, ready‑to‑use
MassRuler™ Express HR Forward DNA Ladder, ready‑to‑use
#SM0393
MassRuler™ Express HR Reverse DNA Ladder, ready‑to‑use
#SM1243 #SM1253
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
ng/20 µl ng/15 µl ng/10 µl ng/5 µl bp
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
Forward Reverse
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
10000 8000 6000 5000 4000 3000 2500 2000 1500
200 160 120 100 80 60 52 40 32
150 120 90 75 60 45 39 30 24
100 80 60 50 40 30 26 20 16
50 40 30 25 20 15 13 10 8
200 140 100
150 105 75
100 70 50
50 35 25
10000 7000 5000
10000 7000 5000
30 40 60
60
45
30
15
3000
3000
40 30
30 22.5
20 15
10 7.5
2000 1500
2000 1500
22.5 30 45
15 20 30
7.5 10 15
100
75
50
25
140 200
105 150
70 100
35 50
9
10µl/lane,8cmlengthgel, 1XTAE,7V/cm,45min
1%TopVision ™Agarose(#R0491) 10µl/lane,8cmlengthgel,1XTAE,7V/cm,30min
MassRuler™ DNA Ladder Mix, ready‑to‑use
MassRuler™ Express Forward DNA Ladder Mix, ready‑to‑use
#SM0403
MassRuler™ Express Reverse DNA Ladder Mix, ready‑to‑use
#SM1283 #SM1293
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
ng/20 µl ng/15 µl ng/10 µl ng/5 µl bp
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl
Forward
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
10000 200 8000 160 6000 120 5000 100 4000 80 3000 60 2500 52 2000 40 1500 32 1031 200 900 180 800 160 700 140 600 120 500 200 400 80 300 60 200 40 100 80
20 16
10µl/lane,8cmlengthgel, 1XTAE,7V/cm,45min
150 120 90 75 60 45 39 30 24 150 135 120 105 90 150 60 45 30
100 80 60 50 40 30 26 20 16 100 90 80 70 60 100 40 30 20
50 40 30 25 20 15 13 10 8 50 45 40 35 30 50 20 15 10
15 12
10 8
5 4
Reverse
200 140 100
150 105 75
100 70 50
50 35 25
10000 7000 5000
10000 7000 5000
30 40 60
22.5 30 45
15 20 30
7.5 10 15
60
45
30
15
3000
40
30
20
10
2000
3000 2000
100 140
75 105
50 70
25 35
30
22.5
15
7.5
1500
1500
200
150
100
50
200
150
100
50
1000
1000
20
15
10
5
140 100
105 75
70 50
35 25
700 500
700 500
40 60
30 45
20 30
10 15
60 40 20
45 30 15
30 20 10
15 10 5
300 200 100
300 200 100
100 140 200
75 105 150
50 70 100
25 35 50
1%TopVision ™Agarose(#R0491) 10µl/lane,8cmlengthgel,1XTAE,7V/cm,30min
Bulkquantitiesandcustomformulationsavailableuponrequest
419
FastRuler™ DNA Ladders, ready‑to‑use (10-10000bp) DNA ladder, ready‑to‑use FastRuler™ Ultra Low Range DNA Ladder FastRuler™ Low Range DNA Ladder FastRuler™ Middle Range DNA Ladder FastRuler™ High Range DNA Ladder Suppliedwith: 6XMassRuler™ DNA Loading Dye 6X Orange DNA Loading Dye
S I S E R O H P O R T C E L E A N D . 9
Concentration, ng/µl
SM1233 SM1103 SM1113 SM1123
22.2 20 20 20
Volume, µl
Applications
2x500
50-333
Loading, µl/lane
Range, bp
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer T4 Polynucleotide Kinase ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free
p.484 p.428 p.430 p.430 p.244 p.469 p.477 p.476
Description FastRuler™DNAladdersarespecically designedforfastsizingandquanticationof double-strandedDNAin48well(or96-well) highthroughputgels,aswellasinconventional agarosegels.Theseladdersaremixturesofve blunt-endchromatography-puriedindividual DNAfragmentsthatareeasilyresolvedina shortseparationdistance(10-20mm)afteran 8-14minrun.Theladdersareespeciallyuseful forelectrophoresisofPCRproducts.FastRuler™ UltraLowRangeDNALadder,ready-to-usecontainsdephosphorylatedDNAfragmentsandis idealfor5’-endlabelingwithT4Polynucleotide Kinase(#EK0031)inaforwardreaction. Features • Fastseparation(8-14min). • Shortseparationdistance(10-20mM). • Sharpbands. • Easy-to-rememberfragmentsizesand quantities. • Ready-to-use–premixedwithloadingdye. • Suppliedwithloadingdyesolutionforsample DNA.
9
3-20
10-200 50-1500 100-5000 500-10000
5
Storage and Loading Buffer (for FastRuler ™ Ultra Low Range) 10mMTris-HCl(pH7.6),10mMEDTA, 0.025%orangeG,0.005%xylenecyanolFF, 10%glycerol. 6X MassRuler ™ DNA Loading Dye 10mMTris-HCl(pH7.6),0.03%bromophenol blue,60%glyceroland60mMEDTA. 6X Orange DNA Loading Dye 10mMTris-HCl(pH7.6),0.15%orangeG, 0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.ConcentrationofeachDNAfragment andofthecompleteladderdeterminedspectrophotometrically.Theabsenceofnucleases conrmedbyappropriatetests. Storage Storeat-20°C(orat4°Corroomtemperature for6months).
FastRuler™ Ultra Low Range DNA Ladder, ready‑to‑use
FastRuler™ Low Range DNA Ladder, ready‑to‑use
#SM1233
#SM1103 200 100 50 20 10
80 80 80 80 124
60 60 60 60 93
40 40 40 40 62
20 20 20 20 31
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl ng/3 µl 1500 80 60 40 20 12 850 80 60 40 20 12 400 80 60 40 20 12 200 80 60 40 20 12
12 12 12 12 19
50
4%TopVision™Agarose(#R0491) 20µl/lane,1XTBE,7V/cm,14min
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis » 7.1.2.DNA/RNA5’-endlabelingby T4 PNK in the forward reaction
420
4.0 2.0 1.0 1.0
Storage and Loading Buffer 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,10%glycerol.
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl ng/3 µl
www.fermentas.com
Number of Agarose, fragments %
1 ml 1 ml
Related Products • • • • • • • •
Cat. #
p.431 p.431 p.433 p.433 p. 380
80
60
40
20
12
2%TopVision™Agarose(#R0491) 20µl/lane,1XTBE,7V/cm,14min
FastRuler™ Middle Range DNA Ladder, ready‑to‑use
FastRuler™ High Range DNA Ladder, ready‑to‑use
#SM1113
#SM1123 bp ng/20 µl ng/15µl ng/10 µl ng/5 µl ng/3 µl
bp ng/20 µl ng/15 µl ng/10 µl ng/5 µl ng/3 µl
5000
80
60
40
20
12
2000
80
60
40
20
12
850
80
60
40
20
12
400
80
60
40
20
12
10000 4000 2000 1000 500
20
12
100 80 60 40 1%TopVision™Agarose(#R0491) 20µl/lane,1XTAE,7V/cm,14min
80 80 80 80 80
60 60 60 60 60
40 40 40 40 40
20 20 20 20 20
12 12 12 12 12
1%TopVision™Agarose(#R0491) 20µl/lane,1XTAE,7V/cm,14min
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
ZipRuler™ Express DNA Ladder Set, ready‑to‑use (100-20000bp) DNA ladder, ready‑to‑use
Cat. #
ZipRuler™ Express DNA Ladder Set ZipRuler™ExpressDNALadder1 ZipRuler™ExpressDNALadder2 Suppliedwith: 6X Orange DNA Loading Dye
SM1373
0.1
2X50
100-200
Loading, µg(µl)/lane
Range, bp
Number of Agarose, fragments %
0.25-0.5 (2.5-5)
100-10000 200-20000
9
5.0
1 ml
Description TheZipRuler™ExpressDNALadderSetcontains twoladders:ZipRuler™ExpressDNALadder1 andZipRuler™ExpressDNALadder2.Bothare mixturesofchromatography-puriedindividual DNAfragments.Thesetisdesignedforfastand accuratesizingofabroadrangeofDNAfragmentsandallowselectrophoresisatdifferent conditions. Forfastseparation,thetwoladdersareloaded intotwodifferentwellsofthesamegel.For longerruns,equalamountsofbothladdersare loadedintothesamegelwell. ZipRuler™ExpressDNALadder1ispremixed withthe6XOrangeDNALoadingDye(orange Gandxylenecyanol),whereasZipRuler ™ ExpressDNALadder2ispremixedwiththe6X MassRuler™DNALoadingDye(bromophenol blue).Whenbothladdersareloadedinthesame gellane,themigrationofDNAismonitoredby threeelectrophoresistrackingdyes(bromophenolblue,xylenecyanolFFandorangeG).
Related Products • • • • •
Concentration, Amount, Applications µg/µl µg
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer Water, nuclease-free
p.484 p.428 p.430 p.430 p.476
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.Preparationofagarosegelsfor DNA electrophoresis » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.7.SeparationofExpressDNAladders indif ferentelectrophoresisconditions
Features • Flexible-canberunintwolanesorcombinedinonelane. • Assembledofchromatographypuried individualDNAfragments. • Ready-to-use–premixedwithaloadingdyefor directloadingandroomtemperaturestorage. • SuppliedwithaloadingdyeforsampleDNA.
p.431 p.431 p.433 p.434
Storage and Loading Buffer (for ZipRuler™ Express DNA Ladder 1) 10mMTris-HCl(pH7.6),10mMEDTA, 0.025%orangeG,0.005%xylenecyanolFF, 10%glycerol. Storage and Loading Buffer (for ZipRuler™ Express DNA Ladder 2) 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,10%glycerol. 6X Orange DNA Loading Dye 10mMTris-HCl(pH7.6),0.15%orangeG, 0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.ConcentrationofeachDNAfragment andofthecompleteladderdeterminedspectrophotometrically.Theabsenceofnucleases conrmedbyappropriatetests. Storage Store-20°C(orat4°Coratroomtemperature forupto6months).
9
ZipRuler™ Express DNA Ladder Set, ready‑to‑use, #SM1373 % ng/0.5 µg bp 8.8 25.6 8.8 8.8 8.0 8.0 16.0 8.0 8.0
1
2
44 10000 128 5000 44 3000 44 2000 40 1200 40 850 80 500 40 300 40 100
20min
bp ng/0.5 µg % 20000 48 7000 48 4000 46 2500 46 1500 152 1000 40 700 40 400 40 200 40
9.6 9.6 9.2 9.2 30.4 8.0 8.0 8.0 8.0
Mix
% ng/0.5 µg bp
8.8 25.6
20min
1%TopVision ™Agarose(#R0491) 5µl/lane,8cmlengthgel,1XTAE,7V/cm,20 min
1
2
44 10000 128 5000
8.8
44
3000
8.8
44
2000
8.0
40
1200
8.0
40
850
16.0
80
500
8.0
40
300
8.0
40
100
40min
bp ng/0.5 µg %
20000 48 7000 48
9.6 9.6
4000
46
9.2
2500
46
9.2
1500 152
30.4
1000
40
8.0
700
40
8.0
400
40
8.0
200
40
8.0
Mix
bp
20000 10000 7000 5000 4000 3000 2500 2000 1500 1200 1000 850 700 500 400 300 200 100
40min
1%TopVision ™Agarose(#R0491) 5µl/lane,8cmlengthgel,1XTAE,7V/cm,40 min
–ZipRuler™Express DNALadder1 2 –ZipRuler™Express DNALadder2 Mix–Ladder1and Ladder2mixedin equalvolumes 1
Bulkquantitiesandcustomformulationsavailableuponrequest
421
O’RangeRuler™ DNA Ladders, ready‑to‑use (10-6000bp) DNA ladder, ready‑to‑use O’RangeRuler™ 5 bp DNA Ladder O’RangeRuler™ 10 bp DNA Ladder O’RangeRuler™ 20 bp DNA Ladder O’RangeRuler™ 50 bp DNA Ladder O’RangeRuler™ 100 bp DNA Ladder O’RangeRuler™ 200 bp DNA Ladder O’RangeRuler™ 500 bp DNA Ladder O’RangeRuler™ 100+500 bp DNA Ladder Suppliedwith: 6X Orange DNA Loading Dye
Cat. #
SM1303 SM1313 SM1323 SM0613 SM0623 SM0633 SM0643
• • • • •
0.1
0.05
50
25
50-100
100
0.5-1 (5-10)
0.25 (5)
SM0653
10-100 10-150 20-300 50-1000 100-1500 200-3000 500-6000
19 15 15 20 15 15 12
5.0 4.5-5.0 3.5-4.0 1.7-2.5 1.7-2.5 0.8-1.2 0.8-1.2
100-6000
32
1.0-1.2
8-10 4-8 4-8 –
1 ml
Related Products
S I S E R O H P O R T C E L E A N D . 9
Concentration, Amount, Loading, Recommended Number of Agarose, PAGE, Applications µg/µl µg µg(µl)/lane range, bp fragments % %
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer Water, nuclease-free
p.484 p.428 p.430 p.430 p.476
Description O’RangeRuler™DNAladdersaredesigned forprecisesizingofPCRproductsandother double-strandedDNAfragmentsinagarose ornon-denaturingpolyacrylamidegels.These stepladdersconsistofligatedbluntendbasic unitrepeatsof10,15,20,50,100,200or 500bp.TheyarenotrecommendedforDNA quantication. O’RangeRuler™DNAladdersaresuppliedwith 6XOrangeDNALoadingDyeforsampleDNA. OrangeGdye(migratesat50bpina1%agarosegel)andcanbeusedtomonitorDNA migrationinagaroseorpolyacrylamidegels.It isthepreferreddyewhenvisualizationofsmall DNAfragmentsisimportant. Features • Stepladdersof5,10,20,50,100,200or 500bpincrements. • Sharpbands • Easy-to-rememberfragmentsizes. • Evenlyspacedreferencebands. • Ready-to-use–premixedwith6XOrange LoadingDye. • SuppliedwithloadingdyeforsampleDNA.
9
Storage and Loading Buffer 10mMTris-HCl(pH7.6),10mMEDTA, 0.025%orangeG,0.005%xylenecyanolFF, 10%glycerol. 6X Orange DNA Loading Dye 10mMTris-HCl(pH7.6),0.15%orangeG, 0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.TheDNAconcentrationofthecomplete ladderdeterminedspectrophotometrically.The absenceofnucleasesconrmedbyappropriate tests. Storage Storeat-20°C(orat4°Corroomtemperature for6months).
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.3. Preparation of gels for PAGE » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis
www.fermentas.com
422
p.431 p.431 p.432 p.433 p.433
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
O’RangeRuler™ 5 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 10 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 20 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 50 bp DNA Ladder, ready‑to‑use
#SM1303
#SM1313
#SM1323
#SM0613
bp
bp
bp
bp
bp
bp
300 150 100 90 80 70 60 50 40
100 100 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5
50 45 40 35 30 25 20
50 45 40 35
15
30 10
25 20
1µg/lane, 10cmlengthgel, 1XTBE,5V/cm,1h
e d i m a l y r c a y l o p % 0 1
15
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5
150
30
100 90 80 70 60 50
20
40 30
10 20
1µg/lane, 10cmlengthgel, 1XTBE,5V/cm,1h
10
e d i m a l y r c a y l o p % 0 1
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 4
80
60 40
40
20 20
0.5µg/lane, 20cmlengthgel, 1XTBE,8V/cm,3h
0.5µg/lane, 20cmlengthgel, 1XTBE,8V/cm,3h
200 180 160 140 120 100 80
60
10
bp
1000
300
200 180 160 140 120 100
1µg/lane, 10cmlengthgel, 1XTBE,5V/cm,1h
bp
e d i m a l y r c a y l o p % 0 1
1000 500 450 400 350
500 450 400 350 300 250
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 2
300 250 200
200 150
150
100 50
0.25µg/lane, 20cmlengthgel, 1XTBE,5V/cm,1.5h
0.5µg/lane, 20cmlengthgel, 1XTBE,8V/cm,3h
e d i m a l y r c a y l o p % 5
100
50
0.25µg/lane, 20cmlengthgel, 1XTAE,8V/cm,3h
O’RangeRuler™ 100 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 200 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 500 bp DNA Ladder, ready‑to‑use
O’RangeRuler™ 100+500 bp DNA Ladder, ready‑to‑use
#SM0623
#SM0633
#SM0643
#SM0653
bp
bp
bp
bp
bp
1500 1000 900 800 700 600
1500 1000 900 800 700 600 500
500 400
400 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 2
300
300 200
200
100
0.25µg/lane, 20cmlengthgel, 1XTBE,5V/cm,1.5h
e d i m a l y r c a y l o p % 5
100
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200
0.25µg/lane, 20cmlengthgel, 1XTAE,7V/cm,1h
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000
500
0.25µg/lane, 20cmlengthgel, 1XTAE,7V/cm,1h
6000 5500 5000 4500 4000 3500 3000 2500 2000
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100
9
0.25µg/lane, 20cmlengthgel, 1XTAE,7V/cm,1h
0.25µg/lane, 20cmlengthgel, 1XTAE,8V/cm,3h
Bulkquantitiesandcustomformulationsavailableuponrequest
423
Conventional Lambda DNA Markers (15-48502bp) DNA marker, marker # Lambda DNA/EcoRI Marker, 1 Lambda DNA/HindIII Marker, 2 Lambda DNA/HindIII Marker, 2, ready‑to‑use Lambda DNA/EcoRI+ HindIII Marker, 3
S I S E R O H P O R T C E L E A N D . 9
Lambda DNA/EcoRI+HindIII Marker, 3, ready‑to‑use Lambda – pUC MIx Marker, 4 Lambda DNA/Eco47I (AvaII) Marker, 13 Lambda DNA/Eco91I (Bst EII) Marker, 15 Lambda DNA/Eco130I (St yI) Marker, 16 Lambda Mix Marker, 19 Lambda DNA/PstI Marker, 24 Suppliedwith: 6X DNA Loading Dye
Amount, µg
SM0281 SM0101 SM0102 SM0103 SM0191 SM0192
0.5
250 (5x50) 250(5x50) 1250(25x50) 250 (5x50) 250(5x50) 1250(25x50)
500 500 2500 500 500 2500
0.5 (1)
SM0193
0.1
250 (5x50)
500
0.5 (5)
SM0291 SM1051 SM0111 SM0161 SM0231 SM0361
0.5 0.5 0.5 0.5 0.5 0.5
50 250 (5x50) 250 (5x50) 250 (5x50) 250 (5x50) 250 (5x50)
100 500 500 500 500 500
0.5 (1) 0.5 (1) 0.5 (1) 0.5 (1) 0.5 (1) 0.5 (1)
0.5 0.1 0.5
Applications, Loading, 0.5 µg/lane µg(µl)/lane
0.5(1)
Range, bp
Number of Agarose, fragments %
3530-21226
6
0.7
125-23130
8
1.0
125-21226
13
1.0
74-19329 23-8126 117-8453 74-19329 1503-48502 15-11501
6 36 14 11 14 29
1.0 1.5 1.0 1.0 0.7 1.5
0.5 (5) 0.5(1)
2 ml / 10 ml
Related Products • • • • • • • • • • • •
Concentration, µg/µl
Cat. #
™
TopVision Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer Klenow Fragment KlenowFragment,exo– dNTP Set Modied dNTPs T4 Polynucleotide Kinase ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free
p.484 p.428 p.430 p.430 p.250 p.251 p.466 p.470 p.244 p.469 p.477 p.476
9
Description ConventionalLambdaDNAmarkersareusedfor sizingoflineardouble-strandedlargeDNAfragmentsinagarosegels.LambdaDNAdigested tocompletionwiththeappropriaterestriction enzyme(s),puriedanddissolvedinstorage buffer.DNAfragmentscontainstickyends. LambdaDNA/HindIIIMarkerandLambda DNA/EcoRI+HindIIIMarkerareavailablein ready-to-useversions–premixedwith6XDNA LoadingDyefordirectloading(afterheating) ontoagarosegels. MarkerssuppliedinTEbuffercanbelabeled radioactivelywithT4PolynucleotideKinase (#EK0031).Alternatively,theycanbelabeled radioactivelyornon-radioactivelywithKlenow Fragment(#EP0051)orKlenowFragment, exo–(#EP0421)bythell-inreaction(withthe exceptionofLambdaDNA/PstIMarker). Features • Sizingandapproximatequanticationoflarge DNAfragments. • Sharpbands. • Ready-to-useversionsarepremixedwith6X DNALoadingDye. • SuppliedwithloadingdyeforsampleDNA.
Storage Buffer (TE buffer) 10mMTris-HCl(pH7.6)and1mMEDTA. Storage and Loading Buffer (for ready‑to‑use markers) 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,0.005%xylene cyanolFFand10%glycerol. 6X DNA Loading Dye 10mMTris-HCl(pH7.6),0.03%bromophenol blue,0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.TheDNAconcentrationofthecomplete markerdeterminedspectrophotometrically.The absenceofnucleasesconrmedbyappropriate tests. Storage Storeat-20°C. Ready-to-use versions can be stored at room temperatureorat4°Cfor6months.
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis » 7.1. DNA/RNA end labeling » 7.1.4.DNA3’-endlabelingbyll-inof 5’-overhangs
www.fermentas.com
424
p.431 p.431 p.433 p.433 p.380 p.380
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
Lambda DNA/EcoRI Marker, 1
Lambda DNA/HindIII M arker, 2
Lambda DNA/EcoRI+HindIII Marker, 3
#SM0281
#SM0101/2/3
#SM0191/2/3
bp ng/0.5 µg bp ng/0.5 µg 21226*218.8
%
% 43.8 23130*238.4 47.7 9416 97.1 19.4 6557 67.6 13.5 4361* 45.0 9.0
7421 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 0
76.5
15.3
5804 5643
59.8 58.2
12.0 11.6
4878
50.3
10.1
3530*
36.4
7.3
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
2322 2027
564
125
23.9 20.9
5.8
1.3
4.8 4.2
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
1.2
0.3
bp ng/0.5 µg
%
21226*218.8
43.8
5148 4973 4268 3530*
53.1 51.3 44.0 36.4
10.6 10.3 8.8 7.3
2027 1904
20.9 19.6
4.2 3.9
1584 1375
16.3 14.2
3.3 2.8
947 831
9.8 8.6
1.95 1.7
564
5.8
1.2
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
0.5µg/lane,20cmlengthgel,1XTAEbuffer,3V/cm, 18h(untilbromophenolbluedyereachedthebottom ofthegel)
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
Lambda – pUC Mix Marker, 4
Lambda DNA/Eco47I (AvaII) Marker, 13
Lambda DNA/Eco91I (BstEII) Marker, 15
#SM0291
#SM1051
#SM0111
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
bp ng/0.5 µg
%
19329*181.5 7743 72.5 5526 52.0 4254* 40.0 3280 45.5 2690 25.0 2322 21.5 1882 17.5
36.3 14.5 10.4 8.0 9.1 5.0 4.3 3.5
1489
14.0
2.8
1150
11.0
2.2
925
8.5
1.7
697
6.5
1.3
421
4.0
0.8
125 bp fragment is not visible and it comprises 0.3%.**
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 1
bp ng/0.5 µg 8126 83.8 6555 67.6 6442 66.4 3676 37.9 2606 26.9 2555 26.3 2134 22.0 2005 20.7 1951 20.1 1611* 16.6 1420 14.6 1284 13.2 985 10.2 974 10.0 894 9.2 597 6.2 590 6.1 513 5.3 511 5.3 433 4.5 398 4.1 345 3.6 310 3.2 308 3.2
% 16.8 13.5 13.3 7.6 5.4 5.3 4.4 4.1 4.0 3.3 2.9 2.6 2.0 2.0 1.8 1.2 1.2 1.1 1.1 0.9 0.8 0.7 0.6 0.6
bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
0.5µg/lane,8cmlengthgel,1XTAE,7V/cm,1h
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
%
8453* 7242 6369 5687* 4822 4324 3675
87.1 74.7 65.7 58.6 49.7 44.6 37.9
17.4 14.9 13.1 11.7 9.9 8.9 7.6
2323 1929
23.9 19.9
4.8 4.0
1371 1264
14.1 13.0
2.8 2.6
702
7.2
1.4
9
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
74 bp fragment is not visible and it comprises 0.1%.**
272 , 242 , 215 , 151, 88 , 73 , 67 , 45 , 42 , 32 , 29 *, 23 bp fragments are not visible and they comprise 2.7%.**
Lambda DNA/Eco130I (StyI) Marker, 16
Lambda Mix Marker, 19
Lambda DNA/PstI M arker, 24
#SM0161
#SM0231
#SM0361
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 1
bp ng/0.5 µg
%
19329*199.3 7743 79.8 6223 64.2 4254* 43.9 3472 35.8 2690 27.7
39.9 16.0 12.8 8.8 7.2 5.5
1882
19.4
3.9
1489
15.3
3.1
925
421
9.5
4.3
1.9
0.9
bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 0
224 ,117 bp fragments are not visible and they comprise 0.7%.**
%
48502 38416 33498 29946 24508 23994 19397* 17053 15004
61 .5 69 .5 60 .5 54 .0 44.5 43.5 35.0 31.0 27.0
1 2.3 1 3.9 1 2.1 1 0.8 8.9 8.7 7.0 6.2 5.4
12220
22.0
4.4
10086 8614* 8271
18.0 15.5 15.0
3.6 3.1 3.0
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 1
0.5µg/lane,8cmlengthgel, 1XTAE,7V/cm,45min
0.5µg/lane,20cmlengthgel,1XTAEbuffer,3V/cm,18h (untilbromophenolbluedyereachedthebottomofthegel)
74 bp fragment is not visible and it comprises 0.2% .**
1503 bp fragment is not visible and it comprises 0.6%.**
bp ng/0.5 µg 11501*118.6 5077 52.3 4749 49.0 4507 46.5 2838 29.3 2556* 26.3 2459 25.3 2443 25.2 2140 22.1 1986 20.5 1700 17.5
% 23.7 10.5 9.8 9.3 5.9 5.3 5.1 5.0 4.4 4.1 3.5
1159 1093
11.9 11.3
2.4 2.3
805
8.3
1.7
514 468 448
5.3 4.8 4.6
1.1 1.0 0.9
339 3.5 0.7 264 2.7 0.5 247 2.5 0.5 0.5µg/lane,8cmlengthgel,1XTAE,7V/cm,1h
216 ,211,200 ,164 ,150 ,94 ,87 ,72 ,15 bp fragments are not visible and they comprise 2.3%.**
* thecohesiveends(the12ntcossiteoflambdaDNA)offragmentsmayannealandformadditionalbands.Thesefragmentscanbeseparatedbyheatingat65°Cfor5minandthencoolingon icefor3min. **theshortestfragments(oblique)arenotvisibleinstandardelectrophoresis.FragmentlengthsarecalculatedusingrespectiveDNAsequence( see pp.411-412).
Bulkquantitiesandcustomformulationsavailableuponrequest
425
Conventional Phage and Plasmid DNA Markers (8-1353bp) Concentration, µg/µl
Amount, µg
SM0271 SM0301 SM0302 SM0303 SM0251 SM0252
0.5
50 50 250(5x50) 50 50 250(5x50)
100 100 500 100 100 500
0.5 (1)
SM0253
0.1
50
100
0.5 (5)
SM0261 SM0121
0.5 0.5
50
100
0.5 (1) 0.5(1)
SM0123
0.1
50
100
pUC19 DNA/MspI (HpaII) Marker, 23
SM0221 SM0222
0.5
50 250(5x50)
100 500
0.5(1)
pUC19 DNA/MspI (HpaII) Marker, 23, ready‑to‑use
SM0223
0.1
50
100
0.5 (5)
DNA marker, marker #
Cat. #
pBR322 DNA/BsuRI (HaeIII) Marker, 5 pUC Mix Marker, 8 pUC Mix Marker, 8, ready‑to‑use FX174
DNA /BsuRI (HaeIII) Marker, 9
FX174
DNA /BsuRI (HaeIII) Marker, 9, ready‑to‑use FX174 DNA /HinfI Marker, 10 pBR322 DNA/AluI Marker, 20 pBR322 DNA/AluI Marker, 20, ready‑to‑use
S I S E R O H P O R T C E L E A N D . 9
9
Suppliedwith: 6X DNA Loading Dye
TopVision™ Agaroses Loading Dyes 50X TAE Buffer 10X TBE Buffer Klenow Fragment KlenowFragment,exo– dNTP Set Modied dNTPs T4 Polynucleotide Kinase ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free
p.484 p.428 p.430 p.430 p.250 p.251 p.466 p.470 p.244 p.469 p.477 p.476
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.3. Preparation of gels for PAGE » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis » 7.1. DNA/RNA end labeling » 7.1.4.DNA3’-endlabelingbyll-inof 5’-overhangs
www.fermentas.com
426
0.1 0.5
0.5(1)
Range, bp
Number of Agarose, PAGE, fragments % %
8-587
22
2.5
19-1118
17
1.7
0.5 (5) 5.0
0.5(1)
0.5 (5)
72-1353
11
1.7
24-726
21
2.5
11-908
17
1.7
–
26-501
13
1.7
5.0
2 ml / 10 ml
Related Products • • • • • • • • • • • •
0.5
Applications, Loading, 0.5 µg/lane µg(µl)/lane
p.431 p.431 p.432
Description ConventionalPhageandPlasmidDNAmarkers areclassicalmolecularweightstandardsused forsizingandapproximatequanticationof lineardouble-strandedDNAfragmentsinagaroseandnon-denaturingpolyacrylamidegels. Themarkersarecomposedofphageorplasmid DNAdigestedtocompletionwiththeappropriaterestrictionenzyme,puriedanddissolvedin storagebuffer.TheDNAfragmentscontainblunt orstickyends,dependingontherestriction enzymeusedforthemarker’spreparation. pUCMix,F X174DNA/BsuRI,pBR322DNA/ AluIandpUC19DNA/MspImarkersareavailableinready-to-useversions–premixedwith 6XDNALoadingDyefordirectloadingonto agaroseandpolyacrylamidegels. Allconventionalversions(suppliedinstorage (TE)buffer)canbelabeledradioactivelywithT4 PolynucleotideKinase(#EK0031).Alternatively pUCMix,F X174DNA/HinfIandpUC19DNA/ MspImarkerscanbelabeledradioactively ornon-radioactivelywithKlenowFragment (#EP0051)orKlenowFragment,exo–(#EP0421) usingthell-inreaction.
Features • SizingandapproximatequanticationofDNA. • Sharpbands. • Ready-to-useversionsarepremixedwith6X DNALoadingDyefordirectloadingandroom temperaturestorage. • SuppliedwithloadingdyeforsampleDNA. Storage Buffer (TEbuffer) 10mMTris-HCl(pH7.6)and1mMEDTA. Storage and Loading Buffer (for ready‑to‑use markers) 10mMTris-HCl(pH7.6),10mMEDTA, 0.005%bromophenolblue,0.005%xylene cyanolFFand10%glycerol. 6X DNA Loading Dye 10mMTris-HCl(pH7.6),0.03%bromophenol blue,0.03%xylenecyanolFF,60%glyceroland 60mMEDTA. Quality Control Testedinappropriategelelectrophoresisapplications.TheDNAconcentrationofthecomplete markerdeterminedspectrophotometrically.The absenceofnucleasesconrmedbyappropriate tests. Storage Storeat-20°C. Ready-to-use versions can be stored at room temperatureorat4°Cfor6months.
p.433 p.433 p.380 p.380
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
pBR322 DNA/BsuRI (HaeIII) Marker, 5
pUC Mix Marker, 8
FX174 DNA/BsuRI (HaeIII)
#SM0271
#SM0301/2/3
#SM0251/2/3
bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 2
587 540 502 458 434 267 234 213 192 184 124 123 104 89 80
67.3 61.9 57.6 52.5 49.8 30.6 26.8 24.4 22.0 21.1 14.2 14.1 11.9 10.2 9.2
%
bp
13.5 12.4 11.5 10.5 10.0 6.1 5.4 4.9 4.4 4.2 2.8 2.8 2.4 2.0 1.8
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
587* 540* 502 458* 434
%
1118 68.75 881 54.2 692 42.55 501 62.2 489 60.7 404 50.15 331 41.1
13.8 10.8 8.5 12.4 12.1 10.0 8.2
124 123 104
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
89 80
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
267 234 213 192 184
e d i m a l y r c a y l o p % 5
bp ng/0.5 µg
242 190 147 111 110 67
404
%
1 353 1 078 872
125.6 100.1 81.0
2 5.1 2 0.0 16.2
603
56.0
11.2
310 281 271 234 194
28.8 26.1 25.2 21.7 18.0
5.8 5.2 5.0 4.3 3.6
118 72
11.0 6.7
2.2 1.3
bp
1353 1078 872 603
331
6.0 4.7 3.7 2.8 2.7
242
8.3
1.7
111 110
190 147
67
e d i m a l y r c a y l o p % 5
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
310* 281* 271* 234 194
118
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
e d i m a l y r c a y l o p % 5
34,34 0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
21,18 ,11,8 bp fragments are not visible and comprise 5.4% .**
692 501* 489*
30.05 23.6 18.25 13.8 13.65
64 57 51
bp ng/0.5 µg
bp 1118 881
Marker, 9
72
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
26 ,19 bp fragments are not visible and comprise 0.9% .**
FX174 DNA/HinfI Marker, 10
pBR322 DNA/AluI Marker, 20
pUC19 DNA/MspI (HpaII) Marker, 23
#SM0261
#SM0121/3
#SM0221/2/3
bp ng/0.5 µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 5 . 2
%
bp
726 67.4 13.5 713 66.2 13.2 553 51.3 10.3 500 46.4 9.3 427 39.6 7.9 417 38.7 7.7 413 38.3 7.7 311 28.9 5.8 249 23.1 4.6 200 18.6 3.7 151 14.0 2.8 140 13.0 2.6 118 11.0 2.2 100 9.3 1.9 82 7.6 1.5 66,666.1,6.11.2,1.2 48 4.5 0.9
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
726 713 553* 500 427* 417* 413* 311 249 200 151 140 118 100
e d i m a l y r c a y l o p % 5
82
bp ng/0.5µg
) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
%
908 659 656 521
104.1 75.6 75.2 59.7
20.8 15.1 15.0 11.9
403
46.2
9.2
281 257 226
32.2 29.5 25.9
6.4 5.9 5.2
100
11.5
2.3
90
10.3
2.1
bp ng/0.5µg
42
0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
40, 24 bp fragment is not visible and comprise 1.9% .**
bp
501* 489* 404 ) 1 9 4 0 R # ( e s o r a g A ™ n o i s i V p o T % 7 . 1
501 489 404 331
93.3 91.0 75.2 61.6
18.7 18.2 15.0 12.3
242 190 147 111 110 67 34 34
45.0 35.4 27.4 20.7 20.5 12.5 6.5 6.5
9.0 7.1 5.5 4.1 4.1 2.5 6.3 6.3
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
0.5µg/lane,8cmlengthgel, 1XTBE,5V/cm,1.5h
66,66 48
%
63 ,57 ,49 ,46 ,19 ,15 ,11 bp fragments are not visible and comprise 5.9% .**
242 190 147
9
111 110
e d i m a l y r c a y l o p % 5
67
34,34 0.5µg/lane,20cmlengthgel, 1XTAE,8V/cm,3h
Note Loadingthe marker in5% polyacrylamidegels isnot recommendedduetoanomalousmigrationof659,656,403 and257fragments.
331
26 bp fragment is not visible and comprises 1.0% .**
* bandsformananomalouspatternonpolyacrylamidegels. **theshortestfragments(oblique)arenotvisibleinstandardelectrophoresis.FragmentlengthsarecalculatedusingrespectiveDNAsequence(
see pp.411-412).
Bulkquantitiesandcustomformulationsavailableuponrequest
427
Loading Dyes Related Products • • • •
™
TopVision Agaroses 50X TAE Buffer 10X TBE Buffer DNA Markers/Ladders
p.484 p.430 p.430 pp.413-426
S I S E R O H P O R T C E L E A N D . 9
Description DNALoadingdyesolutionsareusedtoprepare DNAmarkersandDNAsamplesforloadingon agaroseorpolyacrylamidegels.Theoptimized solutionscontaindifferentdyes(bromophenol blue,xylenecyanolFFororangeG)forvisual trackingofDNAmigrationduringelectrophoresis.Thepresenceofglycerolensuresthatthe DNAformsalayeratthebottomofthewell.The EDTAincludedinthesolutionsbindsdivalent metalionsandinhibitsmetal-dependent nucleases. 6XDNALoadingDye&SDSSolutionis recommendedforagarosegelanalysisofDNA samplesthatcontainhighamountsofDNA bindingproteins.ThepresenceofSDSeliminates DNA-proteininteractionsandpreventsgel-shifts. 2XRNALoadingDyeismainlyusedforpreparationofRNAsamplesforelectrophoresis,butis alsousedfordenaturingDNAelectrophoresis sinceitcontainsthedenaturingagentformamide.
Quality Control TestedforDNAsamplepreparationpriortoagarosegelelectrophoresis.Theabsenceofdeoxyribonucleasesconrmedbyappropriatetests. 2XRNALoadingDyetestedforRNAsample preparationpriortoagarosegelelectrophoresis. Theabsenceofribonucleasesconrmedby appropriatetests. Storage Storeat-20°C(orat4°Corroomtemperature for12months).
100kb BromphenolblueinTBEbuffer BromphenolblueinTAEbuffer 10kb
XylenecyanolFFinTBEbuffer XylenecyanolFFinTAEbuffer
1kb
100bp
9
10bp 0.5
1
1.5
2
2.5 3 3.5 Agarose, %
4
4.5
5
5.5
Figure 9.3. Tracking dye migration in agarose gels.
Protocols and Recommendations » 9.4.PreparationofDNAsamplesfor electrophoresis » 9.5.PreparationofDNAladders/markers for electrophoresis
www.fermentas.com
428
p.433 p.433
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
Loading dye
6X DNA Loading Dye
6X MassRuler™ DNA Loading Dye
6X Orange DNA Loading Dye
6X TriTrack ™ DNA Loading Dye
Cat. #
R0611
R0621
R0631
R1161
6X DNA Loading R1151 Dye & SDS Solution
2X RNA Loading Dye
R0641
Size, Composition ml
Features
Applications
5x1
6X Solution • Two-colortrackingof • 10mMTris DNAmigrationduring • 0.03%bromophenolblue electrophoresis. • 0.03%xylenecyanolFF • NoDNAmaskingduringgel • 60%glycerol visualizationinUVlight. • 60mMEDTA(pH7.6 • EDTAinhibitsmetal adjustedwithNaOH) dependentnucleases.
•Preparationof DNAforloading onagaroseor polyacrylamidegels.
5x1
• One-colortrackingof 6X Solution DNAmigrationduring • 10mMTris electrophoresis. • 0.03%bromophenolblue • NoDNAmaskingduringgel • 60%glycerol visualizationinUVlight. • 60mMEDTA(pH7.6, • EDTAinhibitsmetal adjustedwithNaOH) dependentnucleases.
• Analysisoflarge DNAmolecules. • Preparationof DNAforloading onagaroseor polyacrylamidegels.
5x1
6X Solution • 10mMTris • 0.15%orangeG • 0.03%xylenecyanolFF • 60%glycerol • 60mMEDTA(pH7.6, adjustedwithNaOH)
• Two-colortrackingof • Analysisofsmall DNAmigrationduring DNAmolecules. electrophoresis. • Preparationof • NoDNAmaskingduringgel DNAforloading visualizationinUVlight. onagaroseor • EDTAinhibitsmetal polyacrylamidegels. dependentnucleases.
5x1
6X Solution • Three-colortrackingof • 10mMTris DNAmigrationduring • 0.03%bromophenolblue electrophoresis. • 0.03%xylenecyanolFF • NoDNAmaskingduringgel • 0.15%orangeG visualizationinUVlight. • 60%glycerol • EDTAinhibitsmetal • 60mMEDTA(pH7.6, dependentnucleases. adjustedwithNaOH)
5x1
• AnalysisofDNA • 1%SDSeliminatesDNAsampleswithhigh 6X Solution proteininteractions,prevents amountsofDNA • 0.03%bromophenolblue appearanceofadditional bindingproteins. • 0.03%xylenecyanolFF bandsduetoannealing • Kineticexperiments. • 60%glycerol ofDNAmoleculeswith • DNAagarosegel • 1%SDS cohesiveends. analysisafterDNA • 100mMEDTA(pH7.6, • EDTAinhibitsmetalrestrictiondigestions, adjustedwithTris) dependentnucleases. ligationordephosphorylationreactions.
1
• Preparationof DNAforloading onagaroseor polyacrylamidegels.
• Two-colortrackingofDNA, 2X Solution RNAmigrationduring • Preparationof • 95%formamide electrophoresis. DNAforloadingon • 0.025%SDS • NoDNA,RNAmasking denaturinggels • 0.025%bromophenolblue duringgelvisualizationinUV • Preparationof • 0.025% xylenecyanolFF light. RNAforloadingon • 0.025%ethidiumbromide • DenaturationofDNA,RNA agaroseorpoly• 0.5mMEDTA duetothepresenceof acrylamidegels. formamide.
Migration of dyes (1% agarose, TAE or TBE buffers)
Picture of tracking dyes*
Xylene cyanol FF: TAE:4160bp TBE:3030bp Bromophenol blue: TAE:370bp TBE:220bp
Bromophenol blue: TAE:370bp TBE:220bp
Xylene cyanol FF: TAE:4160bp TBE:3030bp
Orange G: TAE/TBE:<50bp
Xylene cyanol FF: TAE:4160bp TBE:3030bp Bromophenol blue: TAE:370bp TBE:220bp Orange G: TAE/TBE:<50bp
9
Xylene cyanol FF: TAE:4160bp TBE:3030bp Bromophenol blue: TAE:370bp TBE:220bp
Xylene cyanol FF: TAE:4160bp TBE:3030bp Bromophenol blue: TAE:370bp TBE:220bp
* formoredetailedinformationregardingthemigrationratesofdyesinagaroseandpolyacrylamidegels see Tables9.1and9.2onp.431.
Bulkquantitiesandcustomformulationsavailableuponrequest
429
TopVision™ Agarose Agarose
Cat. #
Size
R0491
100g
R0492
500g
R0801
25g
TopVision™ Agarose
TopVision™ Low Melting Point Agarose
Applications
Page
• Analyticalelectrophoresisofnucleicacids. • Preparativeelectrophoresis. • Blottingassays.
484
• In-gelenzymaticprocessingexperiments. • Analyticalelectrophoresisofnucleicacids. • PreparativeelectrophoresisusingAgarase(#EO0461).
484
Related Products
S I S E R O H P O R T C E L E A N D . 9
9
• • • • • • •
50X TAE Buffer 10X TBE Buffer Agarase DNA Markers/Ladders Loading Dyes GeneJET™ Gel Extraction Kit Silica Bead DNA Gel Extraction Kit
p.430 p.430 p.354 pp.413-426 p.428 p.349 p.353
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis p.431 » 9.2.PreparationofagarosegelsforDNAelectrophoresis p.431
Electrophoresis Buffers Buffer
Cat. #
50X TAE Buffer (Tris-acetate-EDTA)
10X TBE Buffer (Tris-borate-EDTA)
B49
B52
Size
1L
1L
1X composition
Application and features
40mMTris 20mMaceticacid 1mMEDTA pHof50XTAE:8.4
• Usefresh1XTAEbothforthegelandfor • Electrophoresisofnucleicacidsinagaroseand theelectrophoresisrun. polyacrylamidegels. • Toprepare1XTAEbuffer,add20mlof • Usedbothasarunningbufferandasagelprepa50XTAEbufferto980mlofdeionized rationbuffer. waterandmixwell. • RecommendedforresolutionofRNAandDNA • TAEbufferhasarelativelylowbuffering fragmentslargerthan1500b(p),forgenomic capacity,thereforeperiodicreplacement DNAandforlargesupercoiledDNA. ofthebufferduringprolongedelectro• Filteredthrougha0.22µmmembrane. phoresisisrecommended.
89mMTris 89mMboricacid 2mMEDTA pHof10XTBE:8.3
• Usefresh1XTBEbothforthegelandfor • Electrophoresisofnucleicacidsinagaroseand theelectrophoresisrun. polyacrylamidegels. • Toprepare1XTBEbuffer,add100mlof • Usedbothasarunningbufferandasagelprepa10XTBEbufferto900mlofdeionized rationbuffer. waterandmixwell. • RecommendedforelectrophoresisofRNAand • Double-strandedlinearnucleicacid DNAfragmentssmallerthan1500b(p). moleculesmigrateabout10%slowerin • Filteredthrougha0.22µmmembrane. TBEbufferthaninTAEbuffer.
Related Products ™
• TopVision Agaroses • DNA Markers/Ladders • Loading Dyes
p.484 pp.413-426 p.428
Quality Control Theabsenceofendo-,exodeoxyribonucleases andribonucleasesconrmedbyappropriate qualitytests.
Usage recommendations
Storage Therearenotimelimitationsforstorageofthe electrophoresisbuffersatroomtemperature. Ifthebufferisstoredatlowertemperatures,a precipitatemayform,whichiseasilydissolved bygentleheating.
Protocols and Recommendations » 9.1.GeneralrecommendationsforDNA electrophoresis » 9.2.PreparationofagarosegelsforDNA electrophoresis » 9.3. Preparation of gels for PAGE
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430
p.431 p.431 p.432
Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS
Protocols and Recommendations 9.1. General recommendations for DNA electrophoresis • Chooseoptimalgelpercentageaccordingto thetablesbelow: Table 9.1. RecommendedagarosegelsforelectrophoreticseparationofDNAfragments. Agarose gel, %
Range of efcient separation, bp
Approximate positions of tracking dyes, bp* Bromophenol blue
Xylene cyanol FF
TBE buffer
TAE buffer
TBE buffer
TAE buffer
0.5
2 00 0-5 00 00
7 50
1 15 0
1 30 00
1 67 00
0.6
1000-20 000
54 0
850
8 820
11 600
0.7
800-12000
410
660
6400
8500
0.8
800-10000
320
530
4830
6500
0.9
600-10000
260
440
3770
5140
1.0
400-8000
220
370
3030
4160
1.2
300-7000
160
275
2070
2890
1.5
200-3000
110
190
1300
1840
2.0
100-2000
65
120
710
1040
3.0
25-1000
30
60
300
460
4.0
10-500
18
40
170
260
5.0
10-300
12
27
105
165
* Positionsofthetrackingdyescanonlybeestimatedapproximately becausethedyefrontmigratesaswideband.
Table 9.2. Recommendedpolyacrylamidegelsfor electrophoreticseparationofDNAfragments(1). Approximate positions of tracking dyes*
Polyacrylamide gel (with BIS at 1:20), % (w/v)
Range of efcient separation
4.0
100-500 b
50 b
230 b
5.0
70-400 b
35 b
130 b
6.0
40-300 b
26 b
105 b
8.0
30-200 b
19 b
75 b
10.0
20-100 b
12 b
55 b
15.0
10-50 b
10 b
40 b
20.0
5-30 b
8b
28 b
1-10 b
6b
20 b
Bromophenol blue
Xylene cyanol FF
Denaturing gels
30.0
Non‑denaturing gels 3.5
1 00 -1 00 0 bp
1 00 b p
4 60 b p
5.0
80-500 bp
65 bp
260 bp
8.0
60-400 bp
45 bp
160 bp
12.0
50-200 bp
20 bp
70 bp
15.0
25-150 bp
15 bp
60 bp
20.0
5-100 bp
12 bp
45 bp
*Pos itionsofthetrackingdyescanonlybeestimatedapproximately becausethedyefrontmigratesaswideband.
• Chooseelectrophoresisconditionsaccording totherecommendationsbelow: Size of the DNA
Voltage
< 1 kb
5-10 V/cm
Buffer TBE
1‑5 kb
4-10V/cm
TAEorTBE
> 5 kb
1-3 V/cm
TAE
Up to 10 kb, fast electrophoresiswith Express DNA ladders
u p to 2 3V /c m
TA E
• UsethesameDNAloadingdye(suppliedwith theDNAladder/marker)forbothsampleDNA andladder/markerDNA. • Ifpossible,loadequalvolumesofthesample DNAandtheladder/markerDNA.Thesample canbedilutedwith1XDNAloadingdye.
• AvoidhighsaltconcentrationsintheDNA samplesasthismaycausebandshiftduring electrophoresis. • Followingelectrophoresis,visualizeDNAby gelstainingin0.5µg/mlethidiumbromide solution,SYBR®GreenIorGelRed.
9.1.1. Recommendations for accurate in‑gel DNA quantication • dNTPs,oligonucleotides,genomicDNA, RNA,NTPsorbuffercomponentscan interferewithspectrophotometricalmeasurementsandmayleadtoinaccuratequanticationofsampleDNA.Inthesecases,itis besttorelyongelquanticationdata. • Forthemostaccuratequantication,use video-densitometryanalysis. • UsethesameDNAloadingdyesolution(suppliedwiththeDNAladder/marker)forboththe sampleDNAandtheladder/markerDNA. • Comparethesamplebandintensitywiththe ladderbandoftheclosestsize. • Ifpossible,adjusttheconcentrationofthe sampletoapproximatelyequalizeitwiththe amountofDNAinthenearestband.
9.2. Preparation of agarose gels for DNA electrophoresis 9.2.1. Non‑denaturing agarose gel electrophoresis • Useaaskofatleastthreetimesgreater volumethanthatofthesolutiontoavoid boilingover. • Usethesame1Xelectrophoresisbufferto preparethegelandtorunelectrophoresis. • Dilute50XTAEBuffer(#B49)or10XTBE Buffer(#B52)toa1Xconcentrationimmediatelybeforeuse. • UseTBEbufferforanalysisofDNAbands smallerthan1500bp.ForlargerDNA,use TAEbuffer. • Forintensiedgelstaining,addethidium bromidetoboththegelandtheelectrophoresisbufferatanal0.5µg/mlconcentration.Alternatively,stainthegelafter electrophoresis. Weargloveswhenhandlingethidiumbromide. • Forreliableanalysisofsupercoiled/relaxed plasmidDNA,ethidiumbromideshouldnot beincludedintheelectrophoresisbuffer orgel.Thegelshouldbestainedonlyafter electrophoresisiscomplete. • Ethidiumbromidestainingandexposureto UVlightmaycauseDNAalterations.Therefore,minimizeUVexposuretoafewseconds ifthepuriedDNAfragmentswillbeusedfor cloning.
• Stainingbeforeelectrophoresiswithsuch intercalatingdyesasSYBRGreenI,GelRed andothersmaycauseabberantmigrationof DNAbandsandDNALadder,thatmaycause mistakesinsizingofDNA.PerformDNA stainingsteponlyaftergelelectrophoresis. Preparation: 1. Weighouttherequiredamountofagarose (#R0491or#R0801)(dependingonthegel percentage)intoanErlenmeyerask. 2. Addtheappropriatevolumeofeither1XTBE or1XTAEbufferandswirltomix. 3. Weightheaskwiththesolution. For high percentage gels (3‑5%):addan excessamountofdistilledwatertoincreasethe weightby10-20%. 4. Boilthemixtureinamicrowaveoven(at mediumpower)untiltheagarosemelts completely;swirltheaskseveraltimes whileboiling.Topreparethehighestquality agarosegelsofanypercentage,anadditional3-5minofboilingaftercompletely meltingtheagaroseisrecommended.Asignicantamountofwaterevaporatesduring thisprocedureandthereforerestoringofthe initialweight(instep5)isrequiredtoobtain thedesiredpercentagegel. 5. Weightheaskagainandifnecessary,add hotdistilledwatertorestoretheinitialweight. For high percentage gels (3‑5%): check(by weighing)thattheexcess10-20%ofwaterhas evaporatedand,ifneeded,continueboilingto removeanyexcess,oraddhotdistilledwaterto restoretheinitialweight. Optional:addethidiumbromidetoanal concentrationof0.5µg/ml.Mixwellandheatfor 1minwithoutboiling. 6. Coolthesolutionto65-70°C.Pourcarefully onacleancastingplatewithanappropriate comb.Ifbubblesappear,pushthemcarefullyawaytothesideswithapipettetip. 7. Solidifythegelforapproximately30min beforeuse.Lowpercentagelowmeltingpoint agarosegelsneedtobesolidiedat4°C. 8. Immersethegelintothedesiredelectrophoresisbuffer.Loadthesamplesontothegel. 9. Runelectrophoresisat5-7V/cmuntilthe bromophenolbluerunsapproximately2/3of thewaydownthegel. 10. Afterelectrophoresisthegelcanbestained byimmersingitintoa0.5µg/mlethidium bromidesolutionfor15-20min,stainedwith GelRed,SYBR®GreenIoranyotherDNA stainingtechnique. Warning. Hotagarosesolutionshouldbe handledverycarefully.
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9.2.2. Alkaline agarose gel electrophoresis
S I S E R O H P O R T C E L E A N D . 9
9
• DoublestrandedDNAladdersarenot recommendedfordenaturingelectrophoresisastheymayformanatypicalpattern. However,thesediscrepanciesarenormally acceptableforanalysisofcDNAorother ssDNAinalkalinegels. • Useaaskofatleastthreetimesgreater volumethanthatofthesolutiontoavoid boilingover. • Weargloveswhenhandlingethidiumbromide. 1. Weighouttherequiredamountofagarose (dependingonthegelpercentage)intoan Erlenmeyerask. 2. Addtheappropriatevolumeofthebuffer (30mMNaCl,2mMEDTA,pH7.5)and swirltomix. 3‑7. Followstepsfromprotocol9.2.1. 8. Immersethegelforatleastonehour intothealkalineelectrophoresisbuffer (30mMNaOH,2mMEDTA). 9. Loadthesamples. 10. Runelectrophoresisat3V/cminalkaline electrophoresisbuffer(30mMNaOH,2mM EDTA)untilthedyerunsapproximately2/3 ofthewaydownthegel. Afterelectrophoresisthegelshouldbe immersedfor30minin100-300mlof 0.5MTris-HClbuffer,pH7.5andthen stainedina0.5µg/mlethidiumbromide solutionfor30min.Ifstainingisnotsufcient,thewholeprocedurecanberepeated. Warning. Hotagarosesolutionshouldbe handledverycarefully.
9.3. Preparation of gels for PAGE 9.3.1. Non‑denaturing PAGE Reagents: • Deionizedwater • 10XTBEBuffer(#B52) • 20%acrylamide/bisacrylamidesolution • TEMED • 10%(w/v)ammoniumpersulfate(APS)inwater • 0.5µg/mlethidiumbromidesolution • Denaturant–freeloadingdyesolutionfor sampleandladderDNA 1. Foranondenaturing5%polyacrylamidegel solutionof40ml,mixthefollowing: 10X TBE Buffer (#B52)
4 ml
20% acrylamide/bisacrylamide
10ml
deionized water
26ml
Caution:acrylamideisaneurotoxin;always weargloves,safetyglasses,andasurgical maskwhenworkingwithacrylamidepowder. 2. Vigorouslyagitatethesolutionfor1minby magneticstirringtoensurecompletemixing. 3. Add48µlofTEMEDandswirltheaskto ensurethatthesolutionisthoroughlymixed.
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4. Immediatelyadd240µloffresh10%(w/v) APSandmixthoroughly. 5. Pourtheacrylamidebetweenthegelplates andinsertthecomb. Clampthecombinplaceatthetopofthe geltoavoidseparationofthegelfromthe platesastheacrylamidepolymerizes.Allow thegeltopolymerizefor30min. Note PolymerizationbeginsassoonasAPSisaddedtothemixture, soallsubsequentstepsmustbeperformedquickly.
6. Removethecombandanybottomspacers fromthegel.Washthegelplatestoclean anyspilledacrylamideandbesurethatthe spacersareproperlyseatedandclean.Fillthe lowerreservoiroftheelectrophoresistankwith 1XTBEbuffer.Initially,placethegelintothe lowertankatanangletoavoidtheformation ofairbubblesbetweentheplatesandthegel bottom.Clampthegelplatestothetopofthe electrophoresistankandlltheupperreservoir with1XTBEsothatthewellsarecovered. 7. Pre-runandwarmthegelforatleast 30minat5V/cm(constantvoltage). 8. Preparetheladdersforelectrophoresisas recommendedintheproductinsertorin Table9.3onp.433. Loadtherecommendedvolumeofthe ladder,premixedwiththeappropriateloadingdyesolution.Usethesameloadingdye forthesampleDNA. 9. Runthegelat5V/cm,takingcaretoavoid excessiveheating.Runthegelforthetimeindicatedinthecerticateofanalysisoftheladder. 10. Stainthegelina0.5µg/mlethidium bromideaqueoussolutionforabout30min. ExaminethegelundertheUVlight.
9.3.2. Denaturing polyacrylamide/ urea gel electrophoresis Reagents: • Deionizedwater • 10XTBEBuffer(#B52) • 20%acrylamide/bisacrylamidesolution • TEMED • 10%(w/v)ammoniumpersulfate(APS)in water • 0.5µg/mlethidiumbromidesolution • UREA 1. Preparedenaturing10%polyacrylamidegel solutionof40ml,mixthefollowing:
3. Add40µlTEMEDandswirltheaskto ensurethoroughmixing. 4. Immediatelyadd400µloffresh10%(w/v) APSandmixthoroughly. 5. Pourtheacrylamidebetweenthegelplates andinsertthecomb. 6. Clampthecombinplaceatthetopofthe geltoavoidseparationofthegelfromthe platesastheacrylamidepolymerizes.Allow thegeltopolymerizefor30min. Note PolymerizationbeginsassoonasAPSisaddedtothemixture, soallsucceedingactionsmustbeperformedpromptly.
Run the gel: 1. Afterpolymerizationiscomplete,removethe combandanybottomspacersfromthegel. Fillthelowerreservoiroftheelectrophoresis tankwith1XTBEbuffer.Initially,placethe gelintothelowertankatanangletoavoid theformationofairbubblesbetweenthe platesandthegelbottom.Clampthegel platestothetopoftheelectrophoresistank andlltheupperreservoirwith1XTBEso thatthewellsarecovered. 2. Pre-runandwarmthegelforatleast 30minat5V/cm(constantvoltage). Note Heatthegel(buffer)duringthewholerunat60-70ºC.
3. Washthewellswith1XTBEbufferto removeUREAandgelpieces. 4. Loadthesamples. 5. Runthegelat6V/cmuntilthelowerdye frontreachesthethreethirdsofthegel.
bp
300 200
300
150
150
100
100
75
75
50
50
25
4 ml
20
20% acrylamide/bisacrylamide
10ml
15
UREA deionized water
to40ml
Caution: acrylamideisaneurotoxin;always weargloves,safetyglasses,andasurgical maskwhenworkingwithacrylamidepowder. 2. Vigorouslyagitatethesolutionbymagnetic stirringtoensurecompletemixingandsolvingofUREApowder.
200
35
10X TBE Buffer (#B52)
19.2g(to8Mnal concentration)
bp
25
1
2
Figure 9.4. Migration of GeneRuler™ Ultra Low Range and Low Range DNA ladders under dena‑ turing conditions. Laddersarepreparedforelectrophoresisusingformamidecontaining2XRNALoadingDye(#R0641)and separatedon10%denaturingPAGEwith8MUREA. 1 –GeneRuler™UltraLowRangeDNALadder(#SM1211) 2 –GeneRuler™LowRangeDNALadder(#SM1191)
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9. DNA ELECTROPHORESIS 6. Soakthegelforabout15minin1XTBEto removetheureapriortostaining. 7. Stainthegelina0.5µg/mlethidiumbromideaqueoussolutionforabout30min. 8. ExaminethegelundertheUVlight. Note DoublestrandedDNAladdersmayformanatypicalpatternin denaturingelectrophoresis.However,GeneRuler™UltraLow RangeandLowRangeDNALadderscanbesuccessfullyused forsizingssDNA( see Fig.9.4).
1. Add1volumeof6XDNALoadingDye& SDSSolutionto5volumesofDNAsample. 2. Mixwellandheatat65°Cfor10minutes. 3. Chillonice,spindownandload. Note Thepreparedsamplecanbestoredat-20ºCandreusedfor electrophoresisafterseveralfreeze-thawcycles.
9.4. Preparation of DNA samples for electrophoresis 9.4.1. Preparation of DNA samples for conventional DNA electrophoresis 6XDNALoadingDye(#R0611),6XMassRuler™ DNALoadingDye(#R0621),6XOrangeDNA LoadingDye(#R0631),6XTriTrack ™DNA LoadingDye(#R1161)areallusedaccordingto belowprotocol: 1. Add1volumeof6XDNAloadingdyeto 5volumesofDNAsample. 2. Mixwell,spindownandload.
9.4.2. Preparation of DNA samples for denaturing polyacrylamide/urea gel electrophoresis Note Usethesameloadingdyesolutionforthesampleandthe ladderDNA.
1. MixtheDNAsamplewithanequalvolume of2XRNALoadingDye(#R0641). 2. Heatat95ºCfor5min. 3. Chillthesampleonicefor3min. 4. Keepsamplesonicewhileloading.
9.4.3. Preparation of DNA samples for denaturing alkaline agarose gel electrophoresis 1. Mix5volumesoftheDNAsampleorladder withonevolumeof6Xalkalineelectrophoresis loadingbuffer(180mMNaOH,6mMEDTA, 18%Ficoll400,0,05%bromcresolgreen). 2. Heatsamplesandladderat70°Cfor5min, thenchillonicefor3minutes.Loadonto thegel.
9.4.4. Preparation of DNA samples containing DNA binding proteins Use6XDNALoadingDye&SDSSolution (#R1151)topreventtheappearanceofadditionalbandsorgelshiftswhenanalyzing: – probesafterDNArestrictiondigestions,ligationordephosphorylationreactions, – DNAsampleswithhighamountsofDNA bindingproteins, – DNAmoleculeswithcohesiveends, – ortostopanenzymaticreactionduring kineticexperiments.
M
1
2
3
4
5
6
7
M
Figure 9.5. The effect of SDS on electrophoresis on resolving band shifts. M –GeneRuler™DNALadderMix(#SM0331) 1 –0.5µgλDNApreparedforloadingwith6XDNA LoadingDye(#R0611) 2 –0.5µgλDNApreparedforloadingwith6XDNA LoadingDye&SDSSolution(#R1151) 3 – 0.5µg λDNAdigestedwithTsoI(#ER1991), preparedforloadingwith6XDNALoadingDye 4 – 0.5µgλDNAdigestedwithTsoI,preparedforloadingwith6XDNALoadingDye&SDSSolution 5 – 0.4µgofthe2DNAfragmentligationmixture priortoligation 6 – 0.4µgofthe2DNAfragmentligationmixture aftertheligation,preparedforloadingwith6X DNALoadingDye 7 – 0.4µgofthe2DNAfragmentligationmixture aftertheligation,preparedforloadingwith6X DNALoadingDye&SDSSolution
9
9.5. Preparation of DNA ladders/markers for electrophoresis Table 9.3. RecommendationsforloadingofDNAladders/markerssuppliedinTEbuffer. Conventional formulation (suppliedinTEbuffer) DNA ladders/markers Technical specications Amount used per 1 mM width of a gel lane Heating
GeneRuler™ DNA ladders
Conventional Lambda DNA markers
Conventional Phage & Plasmid DNA markers
0.1µg(0.2µl)foragarosegel 0.2µg(0.4µl)forPAGE
0.1µg(0.2µl)foragarosegel
0.1µg(0.2µl)foragarosegel 0.2µg(0.4µl)forPAGE
Donotheat
Heat at 65°Cfor5min; chillonicefor3minbeforeuse
Donotheat
1µl(0.5µg) 2µl 9µl
1µl(0.5µg) 2µl 9µl
1µl(0.5µg) 2µl 9µl
I. Loading on agarose gel: DNA ladder/marker loading dye Water, nuclease‑free (#R0581)
Mix gently and load on gel II. Loading on polyacrylamide gel: DNA ladder/marker loading dye Water, nuclease‑free (#R0581)
2µl(1µg) 0.5µl 0.5µl
NotrecommendedforPAGE
2µl(1µg) 0.5µl 0.5µl
Mix gently and load on gel
9.6. Labeling of DNA ladders/markers Forprotocolssee chapter7Molecular Labeling & Detection onp.380.
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9.7. Separation of Express DNA ladders in different electrophoresis conditions Table 9.8. ZipRuler™ExpressDNALadder(#SM1373)separationatvariouselectrophoresisconditions.
Table 9.4. GeneRuler™ExpressDNALadder (#SM1551)separationatvariouselectrophoresis conditions. Duration of 0.8% 1% 1.2% 1.5% 2% 2.5% electro‑ agarose agarose agarose agarose agarose agarose phoresis TAE TBE TAE TBE TAE TBE TAETBE TAE TBE TAE TBE 23 V/cm 5min 10min 15min 20min
Table 9.5. MassRuler™ExpressHRForwardand ReverseDNALadder(#SM1243and#SM1253) separationatvariouselectrophoresisconditions.
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Duration of 0.7% electro‑ agarose phoresis TAE TBE 7V/cm 10min 20min 30min 40min 50min
1% agarose TAE
TBE
1.3% agarose TAE
TBE
1.5% agarose TAE
TBE
Duration of electro‑ phoresis 10 V/cm
0.8% agarose TAE
TBE
1% agarose TAE
TBE
1.2% agarose
1.5% agarose
1.7% agarose
TAE
TAE
TAE
TBE
TBE
TBE
2% agarose TAE
TBE
Ladder Ladder Ladder Ladder Ladder Ladder 1 2 Mix 1 2 Mix 1 2 Mi x 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mi x 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mi x 1 2 Mix
5min 10min 15min 20min 25min 1 –ZipRuler™ExpressDNALadder1. 2 –ZipRuler™ExpressDNALadder2. Mix–Ladder1andLadder2mixedatequalvolumes.
Excellentseparationofallbands. Incompleteseparationofthetwoclosestbands. Noseparation. Lowestbandsrunoffofgel(8cm).
Table 9.6.MassRuler™ExpressLRForwardand ReverseDNALadder(#SM1283and#SM1293) separationatvariouselectrophoresisconditions. Duration of 0.7% 1% 1.3% 1.5% 2% electro‑ agarose agarose agarose agarose agarose phoresis TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE 7 V/cm 10min 20min 30min 40min 50min
Table 9.7. MassRuler™ExpressForwardand ReverseDNALadderMix(#SM1263and#SM1273) separationatvariouselectrophoresisconditions. Duration of 0.7% electro‑ agarose phoresis TAE TBE 7 V/cm 10min 20min 30min 40min 50min
1% agarose TAE
TBE
1.3% agarose TAE
TBE
1.5% agarose TAE
TBE
Reference 1.Sambrook,J.,etal.,MolecularCloning.ALaboratory Manual,ColdSpringHarborLaboratory,ColdSpringHarbor, N.Y.,12.89,5.42,2001.
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9. DNA ELECTROPHORESIS
Troubleshooting Guide DNA electrophoresis problem
1 Low intensity of all or some DNA bands
2 Smeared DNA bands
3 Atypical banding pattern
4 Curved DNA bands
5 DNA remains in the gel well
6 Incorrect quantication data
1.1 Insufcient amount of DNA loaded
2.1 DNA degradation by nucleases
3.1 marker not heated prior to loading
4.1 Gel incompletely immersed in buffer
5.1 Poorly‑formed gel wells
6.1 Different loading condi‑ tions for ladder & sample
1.2 Insufcient or uneven staining
2.2 Incorrect elec‑ trophoresis conditions
3.2 Denatured DNA
4.2 Low sample volume
5.2 Excess DNA loaded
6.2 Incorrect ladder band chosen for quantication
1.3 DNA run off of the gel
2.3 Gel shift effect
3.3 Different loading condi‑ tions for ladder & sample
4.3 Incorrect elec‑ trophoresis conditions
5.3 Contamination of the DNA sample
6.3 Improper quantication method
1.4 DNA diffusion in the gel
2.4 Excess DNA loaded
3.4 Incorrect elec‑ trophoresis conditions
4.4 Bubbles or physical par‑ ticles in wells or in the gel
5.4 Gel shift effect
6.4 Uneven or high background staining of the gel
1.5 DNA masking by tracking dyes
2.5 High salt concentration in samples
3.5 DNA staining before electropho‑ resis
2.6 Poorly formed gel wells
3.6 Atypical migra‑ tion due to DNA sequence or structure
λ
9
6.5 DNA masking by tracking dyes
3.7 Gel shift effect
3.8 High salt concentration in samples
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Table 9.9. TroubleshootingguideforDNAelectrophoresis.
Problem
Possible cause and recommended solution 1.1. Insufcient amount of ladder was loaded. FollowtherecommendationsforloadingdescribedinthecerticateofanalysisoftheDNAladders/markers (~0.1-0.2µgper1mMgellanewidth). 1.2. Insufcient or uneven staining. Followingelectrophoresis,visualizeDNAbystaininginethidiumbromidesolution(nalconcentration 0.5µg/ml)orSYBR®GreenI. Alternatively,iftheDNAwillnotbeusedforcloning,addethidiumbromidetoboththegelandelectrophoresisbufferatanal0.5µg/mlconcentration. Afteralkalineagarosegelelectrophoresisthegelshouldbeimmersedfor30minin300ml0.5MTris-HCl buffer,pH7.5andonlylaterstainedina0.5µg/mlethidiumbromidesolutionfor30min. Afterdenaturingpolyacrylamidegelelectrophoresiswithurea,soakthegelforabout15minutesin1XTBEto removetheureapriortostaining.Stainthegelin0.5µg/mlethidiumbromidein1XTBEsolutionfor15min. Makesurethatthegelisimmersedcompletelyinthestainingsolution.
S I S E R O H P O R T C E L E A N D . 9
1. Low intensity of all or some of the DNA bands
1.3. DNA run off the gel. Performelectrophoresisuntilthebromophenolbluedyepasses2/3(orangeG,4/5)ofthegel.Refertothe tableonp.431formigrationoftrackingdyesindifferentgels. Makesurethattheentiregelisimmersedcompletelyintheelectrophoresisbufferduringtherun.Makesure thatgelandapparatusarepositionedhorizontallyduringtherun. 1.4. DNA diffusion in the gel. Avoidprolongedelectrophoresisorexcessivestaininganddestainingproceduresasthismaycausediffusion ofsmallerDNAfragmentsinthegel. Avoidlongtermstorageofthegelbeforetakingapicture,asthismaycausediffusionofDNAfragmentsand lowbandintensity. 1.5. DNA masking by electrophoresis tracking dyes. Donotexceedtheamountofelectrophoresistrackingdyesusedforsample/ladderpreparation.Usethe loadingdyesolutionssuppliedwitheveryFermentasDNAladder/marker,asthesesolutionscontainamount oftrackingdyeswhichwillnotmaskDNAunderUVlight. PrepareDNAladdersandprobesaccordingtorecommendationsonp.433. 2.1. DNA degradation by nucleases. Usefreshelectrophoresisbuffers,freshlypouredgels,nucleasefreevialsandtipstominimizenuclease contaminationofDNAsolutions.
9
2. Smeared DNA bands
2.2. Incorrect electrophoresis conditions. Preparegelsaccordingtotherecommendationsonp.431,alwaysusethesameelectrophoresisbufferforboth thegelandrunningbuffer. Ensurethatthewholegelisimmersedcompletelyintheelectrophoresisbufferduringtherun. Donotuseanexcessivelyhighvoltageforelectrophoresis.Runthegelsat5-8V/cm.Toincreasetheband sharpness,usealowervoltageforseveralminutesatthebeginningofelectrophoresis. ExcessivelyhighvoltagemayresultingelheatingandDNAdenaturation. Forfastelectrophoresisunderhighvoltage(upto23V/cm)useGeneRuler ™orO’GeneRuler™ExpressDNA ladders(#SM1551/2/3or#SM1563,p.417). Anexcessivelylowvoltageduringtheentirerunmayresultindiffusionofbandsduringelectrophoresis. Tocalculatetheoptimalelectrophoresisconditions(voltage)andtousetherecommendedV/cmvalue (usually5-8V/cm,dependingontheladder)onehasto: –measurethedistancebetweenelectrodes(cathodeandanode)–X,cm –andmultiplythatX,cmvaluebytherecommendedvoltage(Y,V/cm) –theresult(X,cmxrecommendedY,V/cm)isZ–recommendedvoltagetobeapplied. 2.3. Gel shift effect. DNAbindingproteins,suchasligases,phosphatasesorrestrictionenzymesmayalterDNAmigrationongels andcausetheDNAtoremaininthegelwellorgelshifting. LambdaDNAorotherDNAwithlongcomplementaryoverhangsmayannealandmigrateatypically( see p.425). Tocorrectfortheabovementionedeffects,use6XDNALoadingDye&SDSSolution(#R1151,p.429)whichis supplementedwith1%SDStoeliminateDNA-proteininteractionsandtopreventannealingofDNAmoleculesvialongcohesiveends. AlwaysheatthesesampleswithSDSat65°Cfor10min,chillonice,spindownandload. (continuedonnextpage)
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9. DNA ELECTROPHORESIS Table 9.9.TroubleshootingguideforDNAelectrophoresis.
Problem
Possible cause and recommended solution 2.4. Excess DNA loaded. FollowtherecommendationsforloadingdescribedinthecerticateofanalysisoftheDNAladders/markers (~0.1-0.2µgper1mMgellanewidth)orintheTable9.3onp.433.UsesimilarDNAquantitiesforthesamples.
2. Smeared DNA bands
2.5. High salt concentration in the sample. Samplescontaininghighconcentrationsofsaltsmayresultinsmearedorshiftedbandpatterns. Ethanolprecipitationandwashingthepelletwithicecold75%ethanolorspincolumnpuricationpriorto resuspendingthesampleinwaterorTEbufferhelpstoeliminatesaltspresentinthesample. 2.6. Poorly formed (slanted) gel wells. Wheninsertingthecombintothegel,makesurethatitisverticaltothegelsurfaceandstableduringgel castingandsolidication. 3.1. Lambda DNA marker was not heated prior to loading. AllDNAmarkersgeneratedfromLambdaDNA,aswellaslambdaDNAdigestionproductsshouldbeheated at65°Cfor5minandchilledonicebeforeloadingonthegelinordertocompletelydenaturethecohesive ends(the12ntcossiteoflambdaDNA)thatmayannealandformadditionalbands. See p.433forpreparation oflambdamarkersforelectrophoresis. 3.2. Denatured DNA. ExcessivelyhighvoltagemayresultingelheatingandDNAdenaturation. Tocalculatetheoptimalelectrophoresisconditions(voltage)andtousetherecommendedV/cmvalue(often 5-8V/cm,dependingontheladder)onehasto: –measurethedistancebetweenelectrodes(cathodeandanode)–X,cm –andmultiplythatX,cmvaluebytherecommendedvoltage(Y,V/cm) –theresult(X,cmxrecommendedY,V/cm)isZ–recommendedvoltagetobeapplied. Fornon-denaturingelectrophoresisusetheloadingdyesolutionssuppliedwitheveryFermentasDNAladder/ marker,asthesesolutionsdonotcontaindenaturingagents. PrepareDNAladdersandprobesaccordingtorecommendationsonp.433. Donotheatthembeforeloading.HeatingisrequiredonlyforlambdaDNAmarkers.
3. Atypical banding pattern
3.3. Different loading conditions for the sample and ladder DNA. Alwaysusethesameloadingdyesolution(suppliedwiththeDNAladder/marker)forboththesampleDNA andtheladder/markerDNA. Ifpossible,alwaysloadequalorverysimilarvolumesofthesampleDNAandtheladder/markerDNA.The samplecanbedilutedwith1Xloadingdye. 3.4. Incorrect electrophoresis conditions. ExcessiveelectrophoresisruntimesorvoltagemayresultinmigrationofsmallDNAfragmentsoffofthegel. Veryshortorslowelectrophoresismayresultinincompletelyresolvedbands. Rungelsat5-8V/cmuntilthebromophenolbluepasses2/3(orangeG,4/5)ofthegel.Refertothe Table9.1onp.431formigrationoftrackingdyesindifferentgels. Forfastelectrophoresisunderhighvoltage(upto23V/cm)useGeneRuler ™orO’GeneRuler™ExpressDNA ladders(#SM1551/2/3or#SM1563,p.417). TAEbufferisrecommendedforanalysisofDNAfragmentslargerthan1500bpandforsupercoiledDNA. TBEbufferisusedforDNAfragmentssmallerthan1500bpandfordenaturingpolyacrylamidegelelectrophoresis.LargeDNAfragmentswillnotseparatewellinTBEbuffer. ThecorrectgelpercentageisimportantforoptimalseparationoftheladderDNA;preparegelsaccordingto recommendationsonp.431.Whenpreparingagarosegelsalwaysadjustthevolumeofwatertoaccommodate forevaporationduringboiling.Otherwise,thegelpercentagewillbetoohighandresultinbadseparationof largerDNAbands. RefertotheTable9.1onp.431fortherangeofeffectiveseparationofDNAindifferentgels.
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(continuedonnextpage)
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Table 9.9.TroubleshootingguideforDNAelectrophoresis.
Problem
Possible cause and recommended solution 3.5. DNA staining before electrophoresis using uorescent dyes. EthidiumbromideinterfereswithseparationoflargeDNAfragments.Donotincludeethidiumbromideinthe gelandrunbufferwhenlargeDNA(morethan20kb)orsupercoiledDNAisanalyzed.Stainthegelfollowing electrophoresisina0.5µg/mlethidiumbromidesolutionfor30min. StainingbeforeelectrophoresiswithsuchintercalatingdyesasSYBRGreenI,GelRedandothersmaycause abberantmigrationofDNAbandsandDNALadder,thatmaycausemistakesinsizingofDNA.PerformDNA stainingsteponlyaftergelelectrophoresis. 3.6. Atypical migration due to differences in D NA sequence or structure. DuringhighresolutionelectrophoresisDNAfragmentsofequalsizecanmigratedifferentlyduetodifferencesinDNAsequences.ATrichDNAmaymigrateslowerthananequivalentsizeGCrichDNAfragment. ThesequencesofFermentasDNAladdersarechosentoallowforhighlyaccurateDNAmigrationaccording tosize,however,duetodifferenciesinnucleotidesequenceortheoverallDNAstructure,samplemigration cansometimesslightlydifferfromladderbandmigration. DNAstructuressuchasnicked,supercoiledordimericmoleculeswillalwaysshowdifferentmobilityongels comparedtoanequivalentDNAsizestandard. See thepicturebelowformigrationofplasmidDNAforms:
S I S E R O H P O R T C E L E A N D . 9
bp
3. Atypical banding pattern
10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1000 750 500 250
1
2
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1 GeneRuler™1kbDNALadder(#SM0311) 2 UndigestedplasmidpUC192,7kbDNA,forms: – upperband(~4kb)–dimericplasmid – below,lessvisible(~3.5kb)–nickedplasmid – lowestband(~1.9kb)–supercoiledplasmid. 3 LinearizedplasmidpUC19(2,7kb)–migratesaccordingtoitssize
HighlevelDNAmodicationssuchasmethylation,labelingwithbiotinorlargeuorescentmoleculesalso resultinslowermigrationcomparedtounmodiedDNAofthesamesize. 3.7. Gel shift effect. ThepresenceofDNAbindingproteinsinthesample,suchasligases,phosphatasesorrestrictionenzymes mayalterDNAmigrationinthegelorcausetheDNAtoremaininthegelwells. LambdaDNAorotherDNAwithlongcomplementaryoverhangsmayannealresultinginanatypicalmigrationpattern. Toeliminatetheseeffects,use6XDNALoadingDye&SDSSolution(#R1151)whichissupplementedwith 1%SDStoeliminateDNA-proteininteractionsandtopreventannealingofDNAmoleculesvialongcohesiveends. AlwaysheatthesesampleswithSDSat65°Cfor10min,chillonice,spindownandload. Highsaltconcentrationinthesamplemayalsocausegelshifteffects,see below(3.8).
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3.8. High salt concentration in the sample. Sampleswithahighsaltconcentrationmaygivesmearedorshiftedbandpatterns. Ethanolprecipitationandwashingthepelletwithicecold75%ethanolorspincolumnpuricationprior resuspendingDNAinwaterorTEbuffer,helpseliminatesaltfromthesample. (continuedonnextpage)
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Forpatentandlicenseinformationsee p.557
9. DNA ELECTROPHORESIS Table 9.9.TroubleshootingguideforDNAelectrophoresis.
Problem
Possible cause and recommended solution 4.1. Gel incompletely immersed in electrophoresis buffer. Electrophoresisbuffershouldcompletelycovertheentiregelduringsampleloadingandrun. 4.2. Low sample volume. Thesampleortheladdervolumeshouldbelargeenoughtoll1/3ofthetotalcapacityofthewell.Large wellsshouldnotbeusedwithsmallsamplevolumes.Ifneededthesamplevolumecanbeadjustedwith 1Xloadingdye.
4. Curved DNA bands
4.3. Incorrect electrophoresis conditions. Donotuseanexcessivelyhighvoltageforelectrophoresis.Runthegelsat5-8V/cm.Tominimizebandcurving,usealowervoltageforseveralminutesatthebeginningofelectrophoresis. Forfastelectrophoresisunderhighvoltage(upto23V/cm)useGeneRuler ™orO’GeneRuler™ExpressDNA ladders(#SM1551/2/3or#SM1563,p.417). Tocalculatetheoptimalelectrophoresisconditions(voltage)andtousetherecommendedV/cmvalue(which isinmanycases5-8V/cm,dependingontheladder)onehasto: –measurethedistancebetweenelectrodes(cathodeandanode)–X,cm –andmultiplytheXvaluebytherecommendedvoltage(Y,V/cm) –theresult(XxY)istherecommendedvoltagetobeapplied. 4.4. Bubbles or physical particles in the gel wells or in the gel. Usepurewater,cleanasksandcleanequipmentforpreparationofgels. Pourthegelslowlyavoidingformationofbubbles.Bubblescanberemovedwithapipettetip. 5.1. Poorly formed gel wells. Removethegelcombonlyaftercompletepolymerizationofthegel.Pourthebufferontothegelimmediately. Rinsethewellswithelectrophoresisbuffertoremoveureafromdenaturingpolyacrylamidegelspriortoloadingthesample. 5.2. Excess DNA loaded. FollowtherecommendationsforloadingdescribedinthecerticateofanalysisoftheDNAladders/markers (~0.1-0.2µgper1mmgellanewidth)orintheTable9.3onp.433.Ifpossibleloadthesamequantityofthesample.
5. DNA remains in the gel well
5.3. Contamination of the DNA sample. EnsurethatyoursampleDNAsolutiondoesnotcontainanyprecipitate. 5.4. Gel shift effect. ThepresenceofDNAbindingproteinsinthesample,suchasligases,phosphatasesorrestrictionenzymes mayalterDNAmigrationinthegelandcausetheDNAtoremaininthegelwells. LambdaDNAorotherDNAwithlongcomplementaryoverhangsmayannealresultinginanatypicalband migrationpattern. Toeliminatetheseeffects,use6XDNALoadingDye&SDSSolutionwhichissupplementedwith1%SDSto eliminateDNA-proteininteractionsandtopreventannealingofDNAmoleculesvialongcohesiveends. AlwaysheatthesesampleswithSDSat65°Cfor10min,chillonice,spindownandload.
6. Incorrect quantication data
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6.1. Different loading conditions for the sample and the ladder DNA. Alwaysusethesameloadingdyesolution(suppliedwiththeDNAladder/marker)forboththesampleDNA andtheladder/markerDNA. Ifnecessary,adjusttheconcentrationofthesampletoapproximatelyequalizeitwiththeamountofDNAin thenearestband. UseequalorverysimilarvolumesofthesampleDNAandtheladder/markerDNA.Thesamplecanbediluted with1Xloadingdyesolution. 6.2. Incorrect ladder band chosen for quantication of the sample. Alwayscomparethesamplebandwithaladderbandofsimilarsize. 6.3. Improper quantication method used. Ifpossible,quantifybyvideo-densitometrywhilesubtractingthegelbackgroundasthismethodismore precisethanavisualcomparisonofthebands. (continuedonnextpage)
Bulkquantitiesandcustomformulationsavailableuponrequest
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