Oct 25, 2020
PCR (Error-prone PCR)
- Zhujun Wei1
- 12020 iGEM NEFU China
- 2020 iGEM NEFU China
Protocol Citation: Zhujun Wei 2020. PCR (Error-prone PCR). protocols.io https://dx.doi.org/10.17504/protocols.io.bnxrmfm6
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 25, 2020
Last Modified: October 25, 2020
Protocol Integer ID: 43729
Keywords: pcr, prone pcr, error
Safety warnings
Please wear gloves during experiments.Don't touch the lid after PCR program initiation.
Synthesize primers in advance before starting.
Set up a small box with ice, put the tubes of DNA, 2 x Mut Random System, Mut Enhancer and ddH2O into it before loading them into the Bio-rad S1000TM Thermo Cycler.
Add the following reagents to a PCR tube (20 μL).
| Ingredient | Volume | |
| Template Plasmid(1-10 ng/µL) | 0.4 µL | |
| 2 x Mut Random System | 10 µL | |
| Forward Primer (10 μM) | 0.4 µL | |
| Reverse Primer (10 μM) | 0.4 µL | |
| Mut Enhancer | 0-20 µL | |
| ddH2O | To 20 µL |
Program the thermocycler as follows:
| Temperature | Time | |
| 95℃ | 2 min | |
| 94℃ | 30 s | |
| 55℃ | 1 min | |
| 72℃ | 1 min/kilo basepairs | |
| 72℃ | 7 min | |
| 4℃ | ∞ |
Note: Higher initial template concentrationscan lead to lower mutation rates. More amplification cycles may cause higher mutation rates.
Build a mutant library, expand the strains, and add the corresponding inducers, collect data using a Microplate reader operating procedure V.1, and use the GraphPad Prism for data processing and analysis.
Select the positive results for DNA sequencing to determine the mutated nucleotides in the DNA promoter or coding sequence.