Colony PCR is a convenient high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. Individual transformants can either be lysed in water with a short heating step or added directly to the PCR reaction and lysed during the initial heating step.What is the best way to dilute colonies before PCR?
Endeavor to touch only the bacterial colony alone and not the surrounding agar. Dilute your colonies in water or briefly dip them in your PCR reactions. Diluting colonies in water aids troubleshooting down the line and also allows you to make serial dilutions if need be.How much DNA template do I need for a colony PCR?
PCR doesn’t need that much DNA template. Colony PCR works better from liquid cultures than from colonies from plates. This protocol can likely be optimized further. This “protocol” is an outline that you should amend with manufacturer’s instructions and internet searches.Is it possible to do PCR on Bacillus subtilis clones?
It is best to consider the colonies that appear within 48 hours for screening. Good quality and consistent colony PCR of Bacillus subtilis clones is possible. I have used following method for colony PCR: Pick a colony with a pipette tip and mix with 300 uL sterile water.