I still remember my JOY (sadly, yes) when I first learned about colony PCR. It allowed me to avoid a laborious procedure involving genomic DNA isolation, restriction digestion, and (the dreaded) Southern blotting. I was trying to show definitively that a transposon had successfully been inserted into a target gene. Using colony PCR, I could just amplify the DNA from a colony and then show that the transposon increased the size of the expected PCR product compared to a control. Joy. I could also use colony PCR to screen DNA libraries for desired recombinant clones, thus avoiding many minipreps. More Joy. There had to be a catch, it seemed too easy, almost CSI-esque.
Colony PCR is now a widely-used method for quickly screening large numbers of bacterial cells for a gene of interest. When screening recombinant clones, PCR screening of colonies decreases the screening time by one full day compared with miniprep-based methods. Use of a hot-start polymerase in the PCR also allows the convenience of leaving the PCR reactions at room temperature for up to 24 hours before processing, something that becomes attractive when you have a large number of colonies to process or a long walk to fill your ice bucket.
To perform colony PCR, well-isolated colonies are picked using a sterile pipette tip or toothpick, suspended in 100 μl of nuclease-free water, and incubated at 95 °C for 5 minutes. An aliquot of this colony suspension is then added to each PCR reaction. In the reference (1) below, 5 μl of colony suspension was used in a 50 μl amplification with gene-specific primers.
It is also possible to perform colony PCR without this initial step, just by adding master mix to the colony suspension, as described in the video below by Benchfly.
[youtube=http://www.youtube.com/watch?v=h0yRrDWtdA4&hl=en_US&fs=1]
As with all methods, there are variables that need to be optimized for each individual case. For example, colony size and the amount of colony suspension added to reactions can have a significant impact on results, and will need to be determined for the specific target involved. As highlighted in the video, it is also important to keep track of samples by carefully labeling both cultures and PCR tubes, so that the clones containing positive colonies are not lost and can be easily identified later.
Colony PCR References
- Wheeler, S. (2009) Recombinant Clone Screening Using the GoTaq Hot Start Green Master Mix. Promega Notes 100, 13-14.
- Zon, L.I. et al.(1989) The polymerase chain reaction colony miniprep. Biotechniques 7, 696–7.
- Ohno, K. et al. (1991) Direct DNA sequencing from colony: analysis of multiple deletions of mitochondrial genome. Biochem. Biophys. Acta 1090, 9–16