Oct 24, 2020
Colony PCR
- 1Northeast Forest University
- 2020 iGEM NEFU China
Protocol Citation: Zhujun Wei 2020. Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bnwrmfd6
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 24, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 43697
Abstract
This PCR method can be used to screen for inserted target genes or DNA sequencing analysis.
Materials
2×high Taq Master Mix (Enzyme)
Template
F/R primers
ddH2O
Bio-rad S1000TM Thermo Cycler.
Safety warnings
Please wear gloves for the experiment, don't try to touch the lid after PCR program initiation.
Before start
Synthesize primers in advance.
Pick colonies as the template for colony PCR. The number picked for each plate depends on the difference between the positive and negative controls.
| 2×high Taq Master Mix (Enzyme) | 5 μl | |
| Template | 0.4 μl | |
| Forward Primer (10 μM) | 0.4μl | |
| Reverse Primer (10 μM) | 0.4 μl | |
| ddH2O | 3.8 μl |
Fill the rest with water.
Test digest performed and products analysed using agarose gel electrophoresis to confirm if correct construct was present.
Use colony PCR to enlarge colony numbers. Then only the positive clones were mini-prepped.