Colony PCR
NUS iGEM
Abstract
2023 NUS-Singapore iGEM team followed this protocol to perform preliminary screening of plasmid-containing colonies without the need for overnight cell culture. This screening aimed to confirm the correct assembly of DNA fragments during Gibson Assembly or the presence of target DNA fragments in the plasmid. After obtaining the results of the colony PCR, the team could then decide whether specific colonies required overnight culturing. This would allow them to perform plasmid extraction the next day, obtain the fully assembled plasmid, and send it out for sequencing to confirm the final structure.
Steps
Circle the colony of interest.
Prepare the mixture below in a PCR tube, the final volume is 50µL:
| A | B |
|---|---|
| KOD OneTM PCR Master Mix (Blue) | 25uL |
| Forward Primer | 1.5uL |
| Reverse Primer | 1.5uL |
| DI Water | 22uL |
Mix the solution well by vortexing the tubes and then split the solution equally into 5 new PCR tubes (each PCR tube would contain 10µL of the solution).
Use an inoculation loop to inoculate some cells from the colony of interest and dunk the inoculation loop into the PCR tube according to the labels.
Run a PCR with the setting below:
| A | B |
|---|---|
| 98°C | 10s |
| 55°C | 5s |
| 68°C | 1 minute |
| Go to step 1, repeat the cycle 25 times |
After the PCR is complete, proceed to run gel electrophoresis for the samples.
After gel electrophoresis is finished, put the agarose gel onto a UV Sample Tray, next, put it into the Gel Doc EZ System, and then run the gel imaging program on the desktop.
Check if the expected band occurs.
Save the gel image and discard the gel into the biohazard bin.
