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Star Republic: Guide for Biologists

Random PCR mutagenesis

ChangBioscience.com
1. Make the following error-prone 10X PCR buffer:

100 mM Tris-HCl pH8.3
500 mM KCl
70 mM MgCl2
0.1% (w/v) gelatin

2. Make a 100 ml PCR reaction:

10X error-prone PCR Reaction Buffer 10 ml
Forward primer (10 mM, 10 pmol/ml ) 5 ml
Reverse primer (10 mM, 10 pmol/ml ) 5 ml
10 mM MnCl2 3 ml
10 mM dATP and dGTP 2 ml
10 mM dCTP and dTTP 10 ml
PCR DNA Polymerase (5 units/ml) 1 ml
DNA template (10 ng/ml) 1 ml
ddH2O 63 ml
Total volume 100 ml

3. Split the reaction mix into 10 ml aliquots. Run PCR and then pool all PCR products together.

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

5. 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