Error-prone PCR (Random Mutagenesis)
NUS iGEM
Published: 2023-10-09 DOI: 10.17504/protocols.io.rm7vzx57xgx1/v1
Abstract
2023 NUS-Singapore iGEM Team followed this protocol to introduce random mutation in DNA fragments.
Steps
Error-prone PCR (Mutagenesis)
1.
Add the primers, the DNA template, and the following reagents from the50µL PCR sample:
| A | B |
|---|---|
| 10x Mutazyme II (10x Reaction Buffer) | 5μL |
| 40mM dNTP Mix | 1μL |
| DI water | 41.5μL |
| Each Primer (both forward & reverse) | 0.5μL |
| Mutazyme II | 1μL |
| DNA Template | 0.25μL |
2.
Mix the solution well.
3.
Place the sample into the Thermal Cycler and set it with the following conditions:
| A | B | C |
|---|---|---|
| Initial Denaturation | 95°C | 2 minutes |
| Denaturation | 95°C | 1 minute |
| Annealing | 55°C | 1 minute |
| Extension | 72°C | 1 minute |
| Go to step 2, repeat the cycle 44 times | ||
| Extension | 72°C | 10 minutes |
| Finish | 12°C | Infinite Loop |
4.
Upon finishing the PCR steps, add 10µL of DNA loading dye.
5.
Proceeds to the gel electrophoresis to isolate the gene fragment of interest.
