Oct 18, 2019
E. coli and B. subtilis Colony PCR
- 1Heinrich-Heine Universität Düsseldorf
Protocol Citation: Igem Dusseldorf 2019. E. coli and B. subtilis Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.8fhhtj6
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 18, 2019
Last Modified: October 18, 2019
Protocol Integer ID: 28873
Prepare a PCR master mix for 9 reactions, as follows:
| 1x Reaction (Volume; µL) | 9x Reactions (Volume; µL) | ||
| Sterile MilliQ Water | 8 | 72 | |
| Red Taq 2x Master Mix(1.5 mM MgCl2) | 10 | 90 | |
| F Primer (10 µM) | 1 | 9 | |
| R Primer (10 µM) | 1 | 9 | |
| Total Volume | 20 | 180 |
Aliquot 20 µL of the master mix in 8 tubes of a PCR strip.
Transfer cells from a single colony (8 in total) using a sterile P2 pipette tip or inoculating loop
Run thermocycler using the following program (extension for 1.5 kb insert):
| Step | Temp (°C) | Time (mm:ss) | Purpose | |
| 1 | 95 | 3:00 | Initial Denaturation | |
| 25 cycles of Steps 2 to 4: | ||||
| 2 | 95 | 0:20 | Denaturation | |
| 3 | 57 | 0:30 | Annealing | |
| 4 | 72 | 1:00 | Extension | |
| 5 | 72 | 5:00 | Final Extension | |
| 6 | 4 | ∞ | Storage |
Note
Extension time may vary depending on insert size
Run samples on a gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.