Colony PCR for screening transgenic yeast
Anbarasu Karthikaichamy
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Abstract
Colony PCR protocol for screening transgenic yeast
Before start
Prepare 10millimolar (mM) sodium hydroxide (NaOH)
Steps
Pick single yeast colony from the transformation plate and re-streak to a fresh plate with appropriate selection marker.
Incubate the plate in a 30°C incubator for 2 days.
After 2 days, pick around 8 colonies from the re-streaked plate using the smallest micropipette tip or sterile wooden toothpick.
Re-suspend the cells into 20µL of 10millimolar (mM) sodium hydroxide (NaOH) by twirling the pipette tip or wooden toothpick inside the NaoH solution.
Microwave the PCR tubes at the highest power setting for 0h 5m 0s.
Take the PCR tubes out of the microwave oven and briefly spin to pellet down the cells using a bench-top centrifuge.
Without disturbing the pellet, carefully aspirate 2µL out of the tubes and use it as template for PCR.
The PCR reaction mix and the primer annealing temperatures can be adjusted depending on the polymerase and the primer pair used.
