COLONY PCR FOR SEQUENCE VERIFICATION
1. Label the colonies to be screened on the plate.
2. For each colony, take half of the colony using a sterile pipette tip.
3. Scratch off the tip on a PCR tube.
4. Perform the PCR in this tube using the colony as the template for amplifying the gene of interest. Use primers that amplify the whole gene of interest. PCR parameters:
- Set the initial denaturation time to 10 minutes.
- Set the denaturation time during the cycles to 15 seconds.
- Extension: 30 sec/kb.
- Set the final extension time to 8 minutes.
- 28-cycle PCR
- Volume of the reaction = 100 µl.
Alternatively, the sequence verification PCR can also be done using the bacterial pellet instead of a colony. In this case:
1. Take 1 mL of an overnight culture (in a 1.5 mL Eppendorf tube) and centrifuge it at 4°C for 3 min at 8400 X g.
2. Manually discard the supernatant.
3. Scratched off the residual pellet fraction with a sterile pipette tip
4. Perform the PCR in this tube. Same PCR parameters as before.