Nov 21, 2023
Colony PCR (E. coli)
- 1MRC LMS
Protocol Citation: Alan Koh 2023. Colony PCR (E. coli). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69ekrlqe/v1
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: March 16, 2023
Last Modified: November 21, 2023
Protocol Integer ID: 78924
Abstract
Fast and easy PCR to check for cloning inserts. This protocol is not suitable for downstream application of the amplified PCR products.
Set up 1x PCR buffer mix
5X Green or Colorless GoTaq® Flexi Buffer: 10µl (1x)
PCR Nucleotide Mix, 10mM each: 1µl (0.2mM each dNTP)
upstream primer: 2µl (1.0µM)
downstream primer: 2µl (1.0µM)
GoTaq® G2 Flexi DNA Polymerase (5u/µl): 0.25µl (1.25u)
MgCl2: 2ul (1 mM)
Nuclease-Free Water to: 50µl
Note: Can reduce the final volume to 25µl to save reagents.
Using a sterile toothpick or 200µl pipette tips, gently touch a single bacterial colony and dip it into the PCR buffer mix.
PCR reaction
Initial denaturation: 95°C, 2 mins
*Denaturation: 95°C, 1 min
*Annealing: 55°C, 30 sec
*Extension: 73°C, 1 min for every 1kb of amplification
Final extension: 73°C, 5 mins
Storage: 12°C or room temperature depending on when the products will be analysed
*20 to 30 cycles (Generally 20 cycles is more than enough to amplify)
Note: Increasing the annealing temperature will increase the specificity of the PCR reaction.
Important: Run gel electrophoresis immediately as PCR products will normally be degraded due to the presence of DNAse from the lysed E. coli. PCR products not recommended for long term storage.