Oct 18, 2020
Colony PCR
- 1South China University of Technology
Protocol Citation: Jiaxin Li 2020. Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bnihmcb6
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 17, 2020
Last Modified: October 18, 2020
Protocol Integer ID: 43305
Draw grid on clean agar plate (use the appropriate antibiotic). You will use this to inoculate an overnight culture for a positive clone.
Select colonies to pick. Streak portion of colony to numbered sector and place the remainder in a correspondingly numbered PCR tube with 20 µL of autoclaved water.
Heat at 95 °C for 10 minutes.
Note
This can be done in PCR machine
Use 1 µl of reaction for colony PCR (T5 Taq Polymerase)
| Component | Volume/ul | Final Concentration | |
| 2XT5 Super PCR Mix | 5ul | 1x | |
| 10uM Forward Primer | 0.4ul | 0.4uM | |
| 10uM Reverse Primer | 0.4ul | 0.4uM | |
| Template DNA | 1ul | <1ug | |
| ddH2O | Add to 10ul | ||
| Total | 10ul |
| Temperature | time | |
| 98℃ | 03:00 | |
| 98℃ | 00:10 | |
| Tm-5℃ | 00:10 | |
| 72℃ | 20s/kb | |
| 72℃ | 02:00 | |
| 4℃ | ∞ |