Mar 18, 2025
Colony Express - E. Coli colony screening
- 1CRAG
Protocol Citation: Aime Jaskolowski 2025. Colony Express - E. Coli colony screening. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyxzjegx9/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 18, 2025
Last Modified: March 18, 2025
Protocol Integer ID: 124571
Abstract
The “Colony Express” screening method is based on selecting positive clones according to the plasmid size contained in the colonies that grow under selection pressure (antibiotic). This method is useful as a preliminary screening tool because it is simple and inexpensive; however, colony PCR is always the preferred method for selecting positive clones.
Materials
Colony Express Buffer
- NaOH 0.2N
- Sucrose 20% w/v
- SDS 0.5% w/v
- 0.2% w/v Bromophenol Blue
- 30% v/v Glycerol
- Distilled water up to final volume
Agarose
TAE 1X Buffer
- 4.92 g Tris-Base
- 1.14 ml Glacial Acetic Acid
- 2 ml EDTA 0.5M pH8
Procedure
Procedure
Replica plate the individual colonies obtained after the transformation process onto a new LB + antibiotic plate to obtain more starting material. Include a replica of the empty vector used for cloning as a control.
In a 96-well plate, add 40 µl of Colony Express buffer to each well. Using a sterile toothpick, transfer a small sample from each individual colony into the corresponding well. Mix well to homogenize.
Incubate for 10 minutes at room temperature.
Load the samples onto a 0.6% agarose gel (be careful, as the gel will be very fragile!) and run at mid-low voltage (for a large size 15cm x 20cm gel use 100 V)
Analyze the size of the clones by comparing them with the empty vector control.