CELL AND MOLECULAR BIOLOGY LABORATORY PRELIMINARY PERIOD EXPERIMENT 1 Use of Micropipettor and Spectrophotometer Micropipettor
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biology are milliliter (mL) and microliter (uL) Micropipettor is a precision pump with a disposable tip
How to use the Micropipettor 1. The volume of air space in the barrel is adjusted by screwing the plunger further in or out of the piston 2. The volume is displayed on the digital readout 3. Depressing the plunger will displace the specified volume of air from the piston 4. Releasing the plunger creates a vaccum, which will draw an equal volume of fluid into the tip 5. Withdrawn fluid is expelled by depressing the plunger - If there are air bubbles, depress the plunger and withdraw needed amount of volume (again) Types of Micropipettors - Red 1. range is 0.5-1.0 uL 2. tip: 10 uL
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Micropipettor is used to transfer volumes that are less than 1mL (small volumes of DNA and reagents) Measures as little as one microliter (uL), one millionth (10^-6) of a litter 1mL= 0.001L 1uL=0.000001L 1L=1000mL 1L=1000000uL Most useful units of liquid measurement in molecular
- Yellow 3. range is 10-100 uL 4. tip: 100 uL - Blue 5. range is 1000-1000 uL 6. tip: 1000 uL USING THE MICROPIPETTOR: • Rotate the volume adjustor to the desired setting- use specific micropipettor for the specific volume • Firmly get the tip on the end of micropipettor tip = the tip
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color should correspond to the micropipettor color Hold the tube NEARLY at eye level and hold the tube firmly between your thumb and forefinger DO NOT: Pipet with the tube in the test tube rack Have another person hold the tube while pipetting Grasp the tube body not the lid so that there is greater control and AVOID CONTAMINATION OF THE MOUTH OF THE TUBE Hold the micropipettor in your palm and wrap your fingers around the barrel – work the plunger/piston with the thumb (HOLD THE MICROPIPETTOR almost vertical when filling it) Micropipettors have two stops – when only getting the desired volume from the tube: PRESS ONCE SECOND STOP: This will introduce an additional volume of AIR to blow out any solution remaining in the tip When withdrawing liquid, NO AIR SPACE at the very end of the tip: if there is air space: expel the sample back to the tube and coalesce the sample by sharply tapping the tube on the bend top or pulsing it in a microfuge EXPELLING SAMPLE: there is a CAPILLARY ACTION when the tip of pipet touches the inside wall of reaction tube in which the sample will be emptied
- Depress up to second stop for excess fluid • TO PREVENT CROSSCONTAMINATION OF REAGENTS 7. Always add appropriate amounts of single reagent sequentially to all reaction tubes 8. Release each reagent drop onto a new location inside the wall, near the BOTTOM of the reaction tube 9. Use a fresh top for each new reagent 10. Switch tip if contaminated Spectrophotometer
Light comes from a light source (tungsten lamp or delirium) then to a monochromator then llight will pass through the sample solution placed in a cuvette and its wavelength will be in the digital display Types of cuvette for UV light 1. Glass cuvette 2. Plastic cuvette 3. Fused quartz Types of cuvette for visible light 1. Glass cuvette •
Types of Spectrophotometers Simple spectrophotometer - Visible light 11. 380 to 750 nm 12. Light is produced by a tungsten lamp • UV-vis spectrophotometer 13. Second lamp is used: DEUTERIUM – turns a visible light spectrophotometer into a UV-visible unit that can measure from: 190-380 nm 14. Available with a variety of features: scanning, multiple cells, intergral printers, and user interfaces •
ABSORBANCE AND CONCENTRATION OF ABSORBING MOLECULE Absorbance vs Transmittance 1. Absorbance: The amount of light that can be absorbed 2. Transmittance: The amount of light that passes
through the solution as it is not absorbed 3. Absorbance rather than the transmittance is most useful in spectrophotometry 4. If no light is absorbed Absorbance = 0 Transmittance = 100% 5. Each unit in absorbance corresponds with an order of magnitude in the fraction of light transmitted A=1, 10% of the light is transmitted (0.10) 90% is absorbed A=2, 1% of the light is transmitted and 99% is absorbed A=3, 0,1% of light is transmitted and 99,9% is absorbed Formula: Transmittance, T = P / P0 % Transmittance, %T = 100 T Absorbance, A = log10 P0 / P A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T What is the relation of Concentration and Absorbance? 6. Explained by the Beer’s Law Beer’s Law A=ebc
Where A is absorbance (no units, since A = log10 P0 / P) e is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimetres.
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The vortex was used to mix ALL the components of the solution! c. What is the accurate absorbance of Bromophenol blue?
c is the concentration of the compound in solution, expressed in mol L-
7. An increase in the absorbance will also lead to an increase in the concentration (direct relationship) ACCURACY VS PRECISION Linear Regression y=mx + b r= Pearson’s correlation coefficient/correlation coefficient r= value should be near 1 or 1 for the graph to be a linear graph
Accuracy - How close the measured value is to the actual value or true value a. Why was Bromophenol blue (BPB) used in the experiment? 8. BPB was used in the experiment as an indicator 9. Can be used as a pH indicator, color marker or dye b. Why was vortex used?
Precision - How close the value of the measurement is close to the other measured values by the group Why is the analytical balance accurate?
1. The analytical balance is accurate because the density of water is 1.0
C1V1=C2V2
! Bromophenol blue: 1.25g/mL
Analytical Balance EXPERIMENT 2 CELL STAINING TECHNIQUES CELL • Appear transparent in in vivo (under the microscope) • Staining the cells will show specific cellular structures of the cell ex: Using the iodine solution to stain the nucleus of the Allium cepa under the brightfield microscope
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Great precision in quantitative chemical analysis Can give up to 4 decimal places (0.0000g) Water: 1:1 ratio with mL to g DENSITY of water:1
Tetrahymena sp. (or hay infusion in the experiment) • Single-celled protozoan (Protista) • Motile: Have cilia that is used for cellular movement • Commonly found in freshwater ponds
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! Computing for Concentration (c2)
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CELL STAINING Stains used: • Lugol’s iodine, IKI (.15g.mL dH2O)
Methyl green (.01 g/mL, 1% HAc) Nigrosin (.01g/mL dH20)
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1. Lugol’s Iodine It is a solution of elemental iodine and potassium iodide in water. It is a mordant in gram staining. • Neurtral stain • It will always change color Staining: •
Stains polysaccharides in nucleus, vacuoles and cilia.
cell
Structures that are stained: 1. Nucleus 2. Glycogen vacuoles 3. Locomotory organelles (cilia) 2. Methyl Green Methyl green is a basic dye primarily consisting of triphenylmethane. Staining: •
The structure of methyl green has two positive charges, therefore making it a cation. Since DNA is negatively charged, brought about by the presence of the phosphate group, the cation, which is the dye, will bind to the DNA (the anion). Structures that are stained: 1. Nucleus (specifically the micronuclei and macronuclei)
An acidic dye made from the oxidation of aniline, which is an organic compound consisting of phenyl group attached to an amino acid. Aniline is the prototypical aromatic amine. Uses negative staining technique.
Physical Form: Black crystals or powder that is soluble in water and slightly soluble in ethanol Acidic Dye: Easily/readily gives up a hydrogen ion and becomes negatively charged
Negative Staining: Instead of staining the cell, the dye stains the background thus making the outline of the cell surface visible Structures that are stained: •
Cell surface or overall structure of the cell
Lugol’s Iodine Methyl Green Nigrosin
Structures Stained Nucleus, glycogen vacuoles, cilia Nucleus Cell sruface
EXPERIMENT 3 PHAGOCYTOSIS IN TETRAHYMENA sp. Phagocytosis in a Tetrahymena (black: food vacuoles)
Parts (in color) Oral apparatus or cytosome Food vacuole Contractile vacuole Macronucleus Micronucleus Longitudunal microtubule bands
Many food particles cannot easily pass through the cellmembrane of a single-celled organisms through diffusion ! • FOOD PARTICLES SHOULD BE INGESTED by the cells through ENDOCYTOSIS ! Endocytosis! •
A process in which the cell takes in materials from the outside by • ENGULFING • FUSING the food particles with its plasma membrane ! Phagocytosis! • A type of endocytosis which is CELL ENGULFING
Cells ingest large food particles (or smaller cells) ! Pinocytosis ! • A type of endocytosis which is CELL DRINKING • Involves very minute food particles or liquid substances ! •
Food particles used 1. Graphite shavings from pencil (insoluble) 2. India ink, 1% (soluble) ! How does the Tetrahymena take in food? • The Tetrahymena makes use of its CILIA in order to create currents that will move the food particles to its oral cavity • Food will be ingested from its oral cavity ! • CILIA near its oral cavity is specialized for the FEEDING of the Tetrahymena ! • Right side of the oral cavity: Feeding structure called the UNDULATING MEMBRANE (made of cilia) • Left side of the oral cavity: Three smaller ciliary membranellae that curve clockwise toward the cytosome • Food particles will move from the oral cavity to the cytosome • PHAGOCYTOSIS: o When food reaches the cytostome - it is enclosed in a membrane bound food vacuole that
pinches off and migrates through the cell. Graphite shavings 2. Insoluble in water 3. Phagocytosis India Ink • • •
Soluble in water Pinocytosis An increase of concentration in india ink, less food vacuoles
Formalin • Used for fixation • Formaldehyde
EXPERIMENT 4 CELL COUNTING USING THE DYE EXCLUSION PRINCIPLE 1. The DYE EXCLUSION TEST - Used to determine the number of viable cells present in a cell suspension - PRINCIPLE Live cells have an intact cellular membrane which can actively exclude certain dyes 1. Typan blue 2. Eosin 3.Propidium idodide Dead cells do not have such integrity and are stained BLUE
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Consits of TWO chambers One chamber is divided into 9 large squares with the dimension of 1x1mm A cover glass (0.1mm) is supported over these squares TOTAL VOLUME OF EACH SQUARE 1.00mmx0.1mm or 0.1mm^3 or 10^-4cm^3 EACH LARGE SQUARE HAS A VOLUME OF 0.0001mL
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2. Hematocytometer is used in cell counting HEMATOCYTOMETER
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Counting using a hemocytometer Cell count in a hemocytometer depends on: • Even distribution of cells in suspension
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Improper filling of chambers (too much or too little) Representative smaple taken for counting with the pipette and added with no air bubbles Counting cells that are attached to each other or in contact with the enclosing boundaries The total number of chambers counted within the hemocytometer The number of cells counted
Bacteria used: Saccharomyces cerevisiae (cell suspension) • Bacteria used in yeast • Baking, alcohol Counting the cells using the hemacytometer • Clean the hematocymeter and cover slip with 70% ethanol • Use a brightfield microscope • The bacteria must be mixed with 0.3% Trypan blue in 3% formalin (trypan blue, staining the dead cells blue and formalin is for the fixation of the cell) • Only introduce the mixture into one side of the cell chamber (2 chambers) and make sure the mixture covers the ENTIRE GRID IN ONE CONTINUOUS MOTION WITH NO BUBBLES AND FLUID DOES NO OVERFLOW INTO THE GUTTER • Viable cells are transparent Nonviable cells are color blue
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Only count the cells within the squares including those that touch the midline at the top and left If there are more than 50 cells per square, DILUTE the cell suspension with the twice amount of PBS (Phosphate Buffer Saline) originally used Phosphate Buffer Saline pH is 7.4 Balanced salt solution Used for diluting cells Can also be used for washing cells before dissociation, transporting cells or tissues or preparing reagents No calcium, magnesium or phenol red
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ORDINARY GLASSS COVERSLIPS CANNOT be used with the hemacytometer – glass coverslip used: • Using the wrong coverslip can: 1. Distort the cell due to the capillary force of the liquid under the coverslip unless it is measured to the precise amount to fill the space under the coverslip 2. Lead to an inaccurate count of cells •
USE NEUBAUER IMPROVED hematocymeter
Spectrophotometry and Cell counting • • •
Determining the Cell Density – OPTICAL DENSITY Optical density is applicable to all solutions: CELL SUSPENSION Spectrophotmeter 600nm
EXPERIMENT 5 RESTRICTION ENDONUCLEASE DIGESTION OF PLASMID DNA What are endonucleases? • • •
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Reagent blank: NO SUPERNATANT (no cell suspension in the medium EQUATIONS
%viability = #viable cells/ (#nonviable+#viable cells) x100 Cells/mL = average count of cells x dilution factor x 10^4 Dilution Factor = cell suspension + Trypan Blue + Phosphate Buffer Saline / cell suspension volume #doublings = concentration 1) – concentration 2)/ log 2
log(cell log (cell
growth rate = #doublings/time doubling time = 1/growth rate
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restriction
ENZYMES that cleave the sugar-phosphate backbone of DNA at specific sites Only cleave at specific sites In several cases, a given enzyme cuts both strands ofduplex DNA within a stretch of just a few bases FROM BACTERIA, a large majority of restriction enzymes have been isolated and appear to serve a hostdefense role Foreign DNA (from an infected virus) will be chopped up and inactivated (restricted) within the bacterium by the restriction enzymes Hamilton O. Smith and Kent Wilcox were the first to characterize a type II restriction enzyme, HindII, which led to the further development of the recombinant DNA technology.
How are restriction enzymes named? • After their HOST OF ORIGIN Example: 1. EcoRI Isolated from: Escherichia coli (strain RY13) 2. HindIII Isolated from Haemophilus influenzae 3. BamHI
Isolated from: Bacillus amyloliquefaciens
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Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate group This will leave a phosphate group on the 5’ ends and a hydroxyl group on the 3’ ends of both strands A few restriction enzymes will cleave single stranded DNA although at LOW efficiency
Types of Recognition Site Ends • 5’ overhangs an asymmetric cut that produces a shorter 5’ end • •
3’ overhangs similar to a 5’ overhang but the shorter end is observes in the two 3’ ends.
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Blunt End symmetric cuts that produce equal length strands.
pBR322 DNA
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Restriction endonuclease combination: ScaI + Sal I 2 Fragments Lengths: Fragment 1 = 1, 168bp Fragment 2 = 3, 193bp
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3. Restriction encoduclease combination: PstI + BamHI + ScaI • 3 Fragments • Lengths: • -ScaI-BamHI: 892 bp • -
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Artificially created plasmid Circular, double stranded DNA 4361 base pairs Used as an E. coli cloning vector Restriction endonuclease combination: BamHI + EcoRI 2 Fragments Lengths: Fragment 1 = 377bp Fragment 2 = 3, 984bp
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BamHI-PstI: 3, 232 bp -PstI-ScaI: 237 bp
4. Restriction endocuclease combination: Eco RI + Sal I + Pst I • 3 Fragments • Lengths: • Fragment 1: 653 bp • Fragment 2: 2, 956 bp • Fragment 3: 752 bp
When the recognition site for each of the restriction enzymes used in the experiment is known, the base pair sequence in a small region at which the pBR322 DNA is cleaved is therefore determined. This information can be used to study specific regions in the pBR322 DNA. Notes: • In Restriction Digestion, buffer then Restriction Enzyme then DNA • Each Restriction Enzyme has its own APPROPRIATE BUFFER • Restriction enzymes need to be placed first before the buffer as these enzymes work fast when it comes to splicing the DNA sequence AGAROSE GEL ELECTROPHORESIS •
5. Restriction endonuclease enzyme: Bam HI + Sal I + 6. 7. Sca I • 3 Fragments • Lengths: • Sca I - Bam HI: 892 bp • Bam HI - Sal I: 276 bp • Sal I - Sca I: 3,193 bp
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1g of Agarose powder in 100mL 1xTAE buffer 1% AGAROSE GEL Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated
TAE BUFFER • Buffer solution containing a mixture of Tris base, acetic acid and EDTA. I • n molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as
from each other, and a lower percentage gel will help separate larger bands. DNA LADDER • Used to determine the DNA bands (DNA band readings are compared to the DNA Ladder)
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DNA and RNA. It is made up of Tris-acetate buffer, usually at pH 8.0, and EDTA, which sequesters divalent cations.
Why is Ethidium bromide used? • Intercalating agent • Fluorescent tag (Nucleic acid stain) How do you get better resolution of bands? a) running the gel at a lower voltage for a longer period of time b) using a wider gel comb c) loading less DNA into well How do you get better separation of bands? If you have similarly sized bands that are running too close together you can adjust the agarose percentage of the gel to get better separation. A higher percentage agarose gel will help resolve smaller bands
GEL ELECTROPHORESIS RESULTS • CONCENTRATION can affect the mobility of the DNA and may cause it to be more condensed and aggregating thus restricting the movement of DNA • The band will be compared with the DNA Ladder EXPERIMENT 6 Extracting DNA from E.coli - DNA Extraction is the process of isolating DNA from cells by mechanical and biochemical methods - DNA extracted will be used to carryout a downstream of genetic experiments or DNA analyses - DNA extraction is a major procedure in molecular biology and forensic science and an essential technique used by biological and medical scientists DNA Extraction 1. Cells containing DNA will be opened chemically or
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mechanically to release DNA DNA is in a suspension, it will be purified from contaminating cellular components such as lipids in the cell membrane by washing it with SDS or a detergent DNA is precipitated using cold alcohol and resuspended in an appropriate volume
protocol according to their laboratory equipment, reagents and target samples
The purity of DNA is tested using the spectrophotometer by measuring the concentration of DNA The wavelength of nucleic acid is 260 nm while proteins have 280 nm
EDTA Ethanol (70%) SDS (Sodium dodecyl sulfate, 20%) NaCl TE buffer EXPERIMENT 7 Marker Gene Amplification Using Polymerase Chain Reaction - “Replication in a tube” - Discovered by Kary Mullis - Fundamental laboratory equipment of molecular biology - Amplify a particular DNA SEQUENCE to extremely high copy numbers - Researchers may comfigure their PCR
What is needed in PCR master mix? Primers are short pieces of DNA that are made in a laboratory. Since they're custom built, primers can have any sequence of nucleotides you'd like
DNA Polymerase is a naturally occurring complex of proteins whose function is to copy a cell's DNA before it divides in two. When a DNA polymerase molecule bumps into a primer that's base-paired with a longer piece of DNA, it attaches itself near the end of the primer and starts adding nucleotides. (In nature, these primers are made by an enzyme called primase). PCR buffer Provides a suitable chemical environment for optimum activity and stability of the DNA polymerase Taq DNA polymerase Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This polymerase enzymatically assemble s a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNAoligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The Taq polymerase optimum temperature is 70 C. dNTP mix (Deoxynucleotides triphosphate mix) The building-blocks from which the DNA polymerase synthesizes a new DNA strand MgCl2 Generally Mg2+ is used, but Mn2+ can be used for PCR-mediated DNAmutagenesis, as higher
Mn2+ concentration increases the error rate during DNA synthesis[9] Sterile H2O PCR and Thermocycling The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. The cycles in PCR means that it is the temperature changes occurring Steps In PCR 1. Initialization This step consists of heating the reaction to a temperature of 94– 96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. 2. Denaturation This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. 3. Annealing The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should only bind to a perfectly complementary part of the template.
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The primers begin to attach to the DNA template
4. Extension/Elongation The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75– 80 °C,[12][13] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to amplify. 5. Final elongation This single step is occasionally performed at a temperature of 70– 74 °C (this is the temperature needed for optimal activity for most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining singlestranded DNA is fully extended. 6. Final hold This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
EXPERIMENT 8 Extraction and Amplification of Rice Genomic DNA - Rice, Oryza sativa has several varieties such as C-4 and IR-64 - Sinandomeng, Jasmine, Black, Risotto, Basmati
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Different types of rice can be identified by molecular means through marker genes such as the RG100 The amplification process is just the same as in experiment 7
EXPERIMENT 9 Bioinformatics tools for Cell and Molecular Biology - Bioinformatics is the mathematical, statistical and computing methods tool used to solve biological problems using DNA and amino acid sequence and related information - The goal of bioinformatics is to uncover the wealth of biological information hidden in the mass of data and obtain a clearer
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insight into the fundamental biology of organisms The application of computer technology to the management of biological information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied to gene-based drug discovery and development.
Application/Software utilized MEGA (Molecular Evolutionary Genetics) - Application since 1993 - MEGA6 - Uses DNA sequence, protein sequence, evolutionary distance or phylogenetic tree data
