Colony PCR
What is Colony PCR?
The definition of Colony PCR is:
Colony PCR the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest.
Bacterial E.Coli Colony PCR Protocol
Colony PCR Quick Prep Method
Step 1:
Pick a bacterial colony with an autoclaved toothpick or pipette tip and swirl it into 25 μl of TE autoclaved dH2O in an autoclaved microfuge tube. Do not remove the entire colony, and mark the area you picked preferrably number the bottom of the plate (and the microfuge tube).
Step 2:
Heat the mix in a boiling water bath or hot (90-100C) heating block for 2 minutes
Step 3:
Spin sample for 2 minutes high speed in centrifuge.
Step 4:
Transfer 20 μl of the supernatant into a new microfuge tube.
Step 5:
Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.
Yeast Colony PCR Protocol
Take yeast cells which were grown on a plate fresh from the 37C incubator (freshly grown yeast are better)
Take an autoclaved toothpick or pipette tip and touch a bit of the yeast colony. Do not scrape away a whole colony. Suspend it into a standard PCR mastermix.
PCR Cycling Conditions:
First Denaturing Step: 95 °C , 5 min
Denaturing Step: 95 °C, 30 sec
Annealing Step: 50-55 °C, 30 sec
Extension Step: 72 °C, 1min/kb
Repeat Steps 2-4: 34 Times
Final Extension: 72 °C, 3 min
Load the entire PCR reaction onto 1% agarose gel.
Colony PCR - Pubmed Published Research
Contaminating insert degradation by preincubation colony PCR: a method for av... Related Articles
Contaminating insert degradation by preincubation colony PCR: a method for avoiding false positives in transformant screening.
Anal Biochem. 2008 Apr 1;375(1):159-61
Authors: Agrawal V, Roy N
Colony PCR is a convenient alternative to conventional plasmid isolation and restriction digestion for high-throughput screening of recombinant colonies. However, an insert carryover from the ligation mix, adhered to colony or agar plate, generates a substantial number of false positives. To avoid this, a simple single-tube technique involving pre-PCR nuclease incubation has been developed by optimizing a buffer system that provides nuclease action and PCR amplification sequentially. Results presented in this work provide a technique that is amenable for high-throughput screening of recombinant colonies.
PMID: 18164254 [PubMed - indexed for MEDLINE]