Sep 09, 2020
Colony PCR -- CHEM 584
- 1Brigham Young University
Protocol Citation: Ken Christensen 2020. Colony PCR -- CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5xky7n
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: In development
We are still developing and optimizing this protocol
Created: September 09, 2020
Last Modified: September 09, 2020
Protocol Integer ID: 41879
Abstract
A quick way to screen transformants for an insert. This is faster and less expensive than doing a restriction digest.
After you have colonies from a transformation
After you have colonies from a transformation
Draw grid on clean agar plate (use the appropriate antibiotic). You will use this to inoculate an overnight culture for a positive clone.
Select colonies to pick. Streak portion of colony to numbered sector and place the remainder in a correspondingly numbered PCR tube with 50 µL of autoclaved water.
Heat at 95 °C for 10 minutes.** This can be done in PCR machine, a heat block, or a boiling water bath
Spin solution on high for 10 minutes to pellet cellular debris, and remove 4 µL for PCR.
Setup and run the PCR reaction, then assess the results using agarose gel electrophoresis. Be sure to include a positive control (e.g., plasmid) if available. Negative controls are also useful if one is available.