Oct 09, 2023
Error-prone PCR (Random Mutagenesis)
- 1National University of Singapore
Protocol Citation: NUS iGEM 2023. Error-prone PCR (Random Mutagenesis). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx57xgx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2023
Last Modified: October 09, 2023
Protocol Integer ID: 88974
Keywords: Error-prone PCR, PCR, Polymerase Chain Reaction, Mutation, Mutagenesis, Random Mutagenesis
Abstract
2023 NUS-Singapore iGEM Team followed this protocol to introduce random mutation in DNA fragments.
Protocol materials
GeneMorph II Random Mutagenesis KitsAgilent TechnologiesCatalog #200550
Step 1
Safety warnings
Proper lab PPE must be worn at all times.
Error-prone PCR (Mutagenesis)
Error-prone PCR (Mutagenesis)
40m
Add the primers, the DNA template, and the following reagents from theGeneMorph II Random Mutagenesis KitsAgilent TechnologiesCatalog #200550 into a PCR tube to make a 50 µL PCR sample:
| Item | Volume | |
| 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 |
Mix the solution well.
Place the sample into the Thermal Cycler and set it with the following conditions:
| Purpose | Temperature | Duration | |
| 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 | |
Upon finishing the PCR steps, add 10 µL of DNA loading dye.
Proceeds to the gel electrophoresis to isolate the gene fragment of interest.