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Star Republic: Guide for Biologists

PCR

1. A standard 50 ml PCR reaction:

10X PCR Reaction Buffer 5 ml
Forward primer (20 mM, 20 pmol/ml ) 1 ml
Reverse primer (20 mM, 20 pmol/ml ) 1 ml
25 mM MgCl2 3 ml
10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP, dTTP each) 1 ml
PCR DNA Polymerase (1 units/ml) 1 ml
DNA template (20-200 ng/ml) 1 ml
ddH2O 37 ml
Total volume 50 ml

2. For multiple reactions make a master mix without the DNA template. Calculate reagents needed using the Master Mix Calculator of BioToolKit. Mix well by gentle pipetting, since polymerase and dNTP sink to the bottom of the tube. Avoid generating air bubbles, since they will make aliquots less accurate. Aliquot to PCR tubes or plates, and add DNA template, and mix.

3. For some PCR thermocyclers, you may need to overlay 10-50 ml of mineral oil.

4. Spin briefly, and put tubes or plates into a thermocycler.

5. Annealing temperature for each pair of primers should be estimated in advance. See PCR primer design.

6. Program the thermocycler:

Step 1 Denaturation 94 C 5 minutes
Step 2 Denaturation 94 C 30 seconds
Step 3 Annealing x C 45 seconds
Step 4 Extension 72 C 1 minute
Step 5 Repeat step 2-4 29 times
Step 6 Extension 72 C 5 minutes
Step 7 Storage 4 C

7. Check PCR products by running 2-5 ml on 2% agrose gel.

8. Optimization:

  • Design primers with matching melting temperatures.
  • Annealing temperature is usually 4-6 C below the calculated melting temperature. Better results are sometimes achieved when a different annealing temperature is used. To find the optimal annealing temperature, run several PCR reactions with different annealing temperatures. Using a PCR thermocyclers with temperature gradients, optimization can be done in one run.
  • Increase Mg++ concentration increases the PCR sensitivity, but also increases undesirable PCR products. MgSO4 is sometimes used in place of MgCl2.
  • Too high the template concentration may increase the amount of contaminants and reduce efficiency.
  • Excessive dNTP can increase the error rate. Lowering the dNTP (10-50 mM) may reduce error rate.
  • To lower error rate, use DNA polymerases with proof-reading activity (e.g., Tli and Pfu).
  • PCR is a very powerful tool for DNA amplification therefore very small amount of contamination may get amplified. PCR tubes, pipette tips, water etc. should be sterilized and kept out of contact with possible contaminants. Clean the working area before and after preparing a PCR reaction. Labs may reserve a bench space for preparing PCR reactions. Short-wave UV light can be used to destroy DNA in the bench area. Positive displacement pipette aids can also be used to minimize contamination.