Feb 22, 2022
Colony PCR for screening transgenic yeast
- 1University of Illinois at Urbana-Champaign
- GEGC lab UIUC
Protocol Citation: Anbarasu Karthikaichamy 2022. Colony PCR for screening transgenic yeast . protocols.io https://dx.doi.org/10.17504/protocols.io.b5g2q3ye
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: February 22, 2022
Last Modified: July 11, 2023
Protocol Integer ID: 58618
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Abstract
Colony PCR protocol for screening transgenic yeast
Safety warnings
Wear safety equipments when handling sodium hydroxide (NaOH) and microwave.
Before start
Prepare 10 millimolar (mM) sodium hydroxide (NaOH)
Pick single yeast colony from the transformation plate and re-streak to a fresh plate with appropriate selection marker.
Individual yeast colonies on the transformation plate.
Re-streaked (master) plate containing 20 representative colonies from the original transformation plate.
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 20 µL of 10 millimolar (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 00:05:00 .
Note
The tubes will be hot. Care should be taken while handling the tubes after microwaving.
5m
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 2 µL 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.