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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
BASIC POLYMERASE CHAIN REACTION (PCR) INSTRUCTIONS
Basic Polymerase Chain Reaction (PCR) Instructions
The most common problem with PCR is the contamination of reactions with foreign DNA. To avoid this, use PCR-dedicated reagents, PCR-dedicated pipettors with plugged tips, and dedicated areas of workspace for PCR reaction preparation and PCR product analysis. Procedure
1. A typical reaction includes:
1X PCR Buffer
5 ng of DNA Template
100 ng Primer A
100 ng Primer B
1X dNTP mix
2.5 Units of Taq Polymerase
Add sterile ddH2O to a final volume of either 50 μl or 100 μl (see Hint #1)
2. Set up PCR reactions in thin-walled PCR tubes on ice. Add Taq Polymerase last or for a "hot start" (see Hint #2).
3. The reaction is divided into 3 repeated cycles. These are typically:
Denaturation at 95°C for 30 seconds
Annealing at between 50°C to 65°C for 1 minute (see Hint #3)
Extension at 72°C for 1 to 2 minutes
4. The number of cycles in each reaction is typically 25 to 40.
5. Denaturation, Annealing, and Extension times need to be determined per reaction. Values for these are dependent on the type of PCR machine used. These values are typically 30 sec for Denaturation and 1 to 2 min each for Annealing and Extension (see Hint #4)
Nucleotides (10X) 2 mM dGTP
2 mM dCTP
2 mM dATP
2 mM dTTP
Magnesium Buffer 25 mM MgCl2
Try concentrations of 1.5, 3.0, 4.5, 6.0, and 10 mM.
Increasing the Magnesium concentration has the same effect as lowering the annealing temperature.
If necessary, Magnesium can be titrated to obtain an optimal concentration.
Extra Magnesium can be added to the PCR reaction.
If using PCR Buffer, a final Magnesium ion concentration of 1.5 mM will be obtained.
PCR Buffer (10X) 500 mM KCl
100 mM Tris-HCl, pH 8.3
15 mM Magnesium Buffer
This buffer can also contain 0.1% (w/v) Gelatin
Primers Each primer should be used at approximately 100 ng per reaction.
Resuspend oligonucleotides in ddH2O at 100 ng/μl.
DNA Template Use between 1 and 5 ng of cloned DNA or between 40 and 100 ng of genomic DNA per reaction.
It is convenient to dilute template stocks to an appropriate concentration (e.g., 5 ng/μl in ddH2O for cloned DNA).
Paraffin Oil or Mineral Oil If the thermocycler has a heated cover, no oil is needed.
This prevents the evaporation of the sample.
In some thermocycler instruments, either Paraffin or Mineral Oil must be overlayed on the PCR volumes.
BioReagents and Chemicals
DNA Polymerase, Taq
1. The amount of sterile ddH2O to add depends on the total amount of product desired.
2. A Denaturation step is included prior to the start of the reaction cycles. This denaturation is usually a 4 min step at 95°C. In a "hot start" PCR reaction, the Taq Polymerase is added to the reaction tubes immediately after this initial Denaturation step. This is to prevent formation of inappropriate products or excessive primer dimers that might occur before the first denaturing, annealing, and extension cycle.
3. The appropriate Annealing temperature must be determined for each pair of primers and template. Reactions should be carried out at a number of different annealing temperatures to determine an optimum value. Start with an annealing temperature that is 5°C below the calculated melting temperature of the primers.
4. Longer PCR products (greater than 1 kb) need longer extension times.