Restriction Endonuclease Digestion of Plasmid DNA J.P. J.P. Latonio, R.E. R.E. Loquellano, Loquellano, N. Lustre, Lustre, M.M. Manalo, Manalo, E. Marasigan, Marasigan, and and F. F. Marzan 4Bio5, College of Siene, !ni"ersit# of Santo $o%as, Es&a'a, Manila
Summary Keywords:
Restrition Restrition endonulease, &BR()), et*idiu% +ro%ide, agarose gel eletro&*oresis
Restriction endonucleases are enzymes that cleave the sugar-phosphate backbone of DNA, they are mostly isolated in bacterias, wherein they also act as defense mechanism for the organism. or this e!periment, the restriction enzymes used are "am#$, %coR$, #ind$$$, &st$, 'ca$, 'al$, 'al $, 'ap 'ap$, $, 'ty 'ty$. $. $n the res restri tricti ction on dig digest estion ion of the pla plasmi smid, d, fiv fivee combinations of restriction enzymes were used and incubated in a dry block heater before adding gel-loading gel-loading dye and loaded separately separately in () agarose gel. *o visualize the sizes of the fragments cleaved during the plasmid digestion, samples were electrophoresed with ethidium bromide as their staining medium. +f the eight wells loaded with samples for e!perimental values, only lanes , , and showed bands under und er /0 lig light. ht. *h *hese ese lan lanes es onl only y e!h e!hibi ibited ted a sin singl glee ban band, d, whi which ch represents the cleaved fragment from one of the enzymes at the same time indicating an error of the differences between the displayed bands with the number of e!pected bands. 1anes (-2 did not display any bands at all which may be due to DNA degradation or DNA denaturation from nuclease contamination and e!cessive heat e!posure, respectiv respe ctively ely.. *he fragment sizes of the cleaved segm segments ents were not determined as well due to the DNA ladder bands lacking inaccuracy.
Introduction Restriction enzym zymes are DNA-cu -cutting enzymes enzymes found found in bacter bacteria. ia. "ecaus "ecausee they cut within the molecule, they are often called called restricti restriction on endonucl endonucleases. eases. A restrictio restriction n enzy enzyme me recog recogni nize zess and and cuts cuts DNA DNA only only at a particular se3uence of nucleotides called its recognition site. *he rarer the site it recognizes, the smaller the number of pieces produced by a given given restri restricti ction on endonu endonucle clease ase.. 'ome 'ome of the
most most common common restri restricit cition ion endonu endonucle clease asess are "am#$ from Bacillus from Bacillus amyloliquefaciens, %coR$ from Escherichia coli and 'all from Strepto Streptomyces myces albus. albus. Each of these these restri restrictio ction n endonucleases have their own unique recognition sites but enzymes called isoschizo isoschizomers mers can have the same recognit recognition ion site. p"R455 is a plasmid DNA isolated isolated from %. coli. *his molecule is a double stranded circle having 6,4( base pairs. *he plasmid p"R455 was one
of the first %75 multipurpose cloning vectors to be designed and constructed 8(9: for the efficient cloning and selection of recombinant DNA molecules in %scherichia coli 8"albas, et al., (9:. Agarose gel electrophoresis is the standard lab procedure for separating DNA by size 8e.g. length in base pairs: for visualization and purification. %lectrophoresis uses an electrical field to move the negatively charged DNA toward a positive electrode through an agarose gel matri!. *he gel matri! allows shorter DNA fragments to migrate more 3uickly than larger ones. *hus, the length of a DNA segment can be accurately determined by running it on an agarose gel alongside a DNA ladder which is a collection of DNA fragments of known lengths.
the wells from left to right starting with the DNA ladder on lane ( and the sample without restriction endonuclease on the e!treme right. *he electrophoresis apparatus was covered with the anode on the side of the wells. *he electrodes were attached to the power supply set at (;;0 52;mA 2;>. >hen the gel loading dye moved up to one half, the power supply was turned off. *he gel was removed gently from the electrophoresis apparatus and was transferred to the developing tray containing (;u1 ethidium bromide per (;;m1 buffer. *he gel was then transferred to the gel documentation system. R f values were measured for all the samples and the digital images of the gel were recorded. 1astly, the gel was immersed in hypochlorite solution overnight before it was discarded.
Results and discussion Ladder
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Materials and Methods Restriction Digestion Distilled water, (;! buffer, restriction endonuclease and p"R455 DNA were added to each of the five (.2m1 microcentrifuge tubes. *o a separate microcentrifuge tube, all the reagents were added e!cept for the restriction endonuclease. Different combinations of restriction endonuclease were assigned per group. *he mi!ture was then incubated for one hour at 4<= in a dry block heater. After incubation, 5 u1 gel loading dye was added to each mi!ture and was loaded separately in () agarose gel. 2;bp DNA ladder was placed in lane ( and electrophoresis was performed at (;;0 52;mA 2;>. Agarose Gel Electrophoresis +ne gram agarose powder was dissolved in (;; m1 (! *A% buffer in a microwave oven. *he gel was cooled to appro!imately ; <= and was poured in the gel casting tray. *he comb was placed in position and the gel was solidified. *he comb was then removed gently. *he gel casting tray was placed into the submarine gel electrophoresis system. *he solidified gel was covered with *A% buffer up to the ma!imum mark, which is a few mm from the upper surface of the gel. *he samples were then loaded into
Figure *he results of the gel are shown in igure (. *he number of restriction enzymes added to the microcentrifuge tubes corresponds to the number of e!pected bands to be seen, which then signifies the number of segments formed after the cleavage. $n the figure shown, only lanes , and showed bands, which confirms that different enzymes cleaved the plasmid, although the e!pected results were not achieved. $deally, lane should have two bands representing the two segments cleaved by the two enzymes namely "am #$ and %co R$ as well as lane which should have two bands
showing fragments cleaved by 'ca $ and 'al $ and lane should have three bands because of the three enzymes? &st $, "am #$ and 'ca $. *he first well is reserved for the DNA ladder that serves as a visual reference for the determination of fragment sizes formed by the restriction enzymes. %ach band represents a number corresponding to the length of segments according to base pairs. $n the results shown still in igure (, bands in the DNA ladder are accumulated at one portion of the gel making it difficult for the bands found on the succeeding wells to be compared with for their sizes. "ands from lanes from (-2 are not observable on the same figure. +ne known error that contributes to this is the insufficiency of 3uantity or concentration of DNA loaded on the gel. Another sources of error could be nuclease contamination of the microcentrifuge tubes leading to DNA degradation and high heat standards e!posure that may cause DNA denaturation.
Figure )
*he plasmid map of p"R455 in igure 5 indicates the restriction sites 8displayed in number: of the different enzymes. *his can be
used to measure the size of DNA fragments cleaved by a particular restriction enzyme. *here are a total of 6,4( base pairs in p"R455 and therefore all calculated segment lengths in base pairs based on the combination of enzymes should sum up to 6,4(.
Acknowledgements Acknowledgement? *he group is thankful to @s. Asia Abdulmaid, Asst. &rof. Bosefino =astillo C Asst. &rof. @ichael "ahrami-#essari for guiding us in doing the e!periment.
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