Colony PCR and colony rescue plate
- For each colony you want to analyze, put 10 uL sterile MQ water in a 1.5 mL test tube
- Using the 2 uL Poseidon pipette, pick white colonies and suspend bacteria in the water
(You do not have to label the colonies on the plate but you must label the tubes.)
Set up the colony PCR Master mix on ice (multiply the volumes shown below by the amount of
colonies you want to test and add 10% to make sure you have enough Master mix):
Per reaction, you will need:
13.5 uL MQ water
1 uL 10x buffer (home-made)
0.5 uL dNTP mix
0.5 uL Primer 5’ (e.g. M13F if using pGEM-TEasy)
0.5 uL Primer 3’ (e.g. M13R if using pGEM-TEasy)
0.2 uL Taq polymerase (home-made)
Do a hot-start PCR!.
Prepare your PCR reactions in 0.2 mL PCR tubes (single or strip tubes):
- Add 2 uL of suspended bacteria to each PCR tube on ice
- Add 18 uL PCR Master mix, and close the lids, vortex, spin down, put on ice
- Put your PCR tubes in the PCR machine and resume the PCR cycle
Backing up your colonies for future Mini prep cultures:
Do not go back to the original plate to get bacteria! Instead:
- Store the suspended bacteria in the fridge (they will last a few days)
- Additionally, spot all bacterial strains side by side (~0.1 uL each) on an agar plate with the
correct antibiotic and incubate o/n at 37oC