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 |
|
| 