Oct 11, 2022
- 1Fudan University
Protocol Citation: Team Fudan iGEM 2022. Colony PCR for 2022 iGEM. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb352vx1/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 10, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71102
Abstract
Based on manual included in Vazyme Taq product
Select colonies on the agar plate to pick. Culture portion of the colony in LB media with antibiotic and grow in 37 degree. Put the remainder of the colony into a correspondingly numbered PCR tube with 20 µL of reaction mix (as below), and pipetting to complete resuspend the bacteria.
| A | B | |
| Component | Volume/ul | |
| 10x Taq Buffer | 2 uL | |
| 10 uM Forward Primer | 0.5 uL | |
| 10 uM Reverse Primer | 0.5 uL | |
| Template DNA (bacteria colony) | added later | |
| dNTP 10 mM mix | 0.5 uL | |
| Taq DNA polymerase | 0.5 uL | |
| distilled water | 16 uL | |
| Total | 20 uL |
Prepare PCR reaction mix before step 1, and place the mix on ice.
Primers used for screening crt genes insertion are:
> 5-crtB
5-ATGAATAATCCGTCGTTACTCAATCATGC-3
> 5-crtE
5-ATGACGGTCTGCGCAAAAAAAC-3
> 5-crtI
5-ATGAAACCAACTACGGTAATTGGTGC-3
> 5-crtY
5-ATGCAACCGCATTATGATCTGATTC-3
> rev320crtB
5-CCTTCCAGATGATCAAACGCGTAAG-3
> rev320crtE
5-ATGAGAATGAATGGTAGGGCGTC-3
> rev320crtI
5-GGATTAAACTGCTGAATCTGCGCTTC-3
> rev320crtY
5-CCGCGGTATCCATCCACAAG-3
5m
| A | B | |
| Temperature | Time | |
| 94℃ | 05:00 | |
| 94℃ | 00:30 | |
| 60℃ | 00:30 | |
| 72℃ | 03:30, cycle 3-4-5 for 25 times | |
| 72℃ | 05:00 | |
| 16℃ | ∞ |
The PCR program above is for screening crt genes insertion during 2022 iGEM season.
10m
Agarose gel electrophoresis of PCR products. One lane on the gel is loaded with 6 uL D2000 DNA ladder. Run the gel at 100 V for 20-30 minutes, and examine the gel under UV.
The correct bacterial clones are sent for Sanger sequencing. Once verified, these clones would be used for further experiments.