Promega
GoTaq Hot Start Colourless Kit
M5132
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E. coli DH5 alpha cells transformed with a plasmid called pCV20 which expressed our gene of interest were screened using primers specific to gene of interest with plasmid backbone. Colony PCR was screened using GoTaq Hot Start PCR ready-to-use master mix kit and successful in getting desired PCR product confirming the presence of the pCV20.
Colony PCR Screening of bacterial colonies
Individual Colony resuspended in nuclease free water
It is better to prepare your PCR template by picking a colony and resuspending it in the water, boiling it for 5 minutes at 100 degree Celsius and spin it, use supernatant for PCR.
Using GoTaq Hot Start PCR ready-to-use kit, I have screened 10 colonies to detect presence of pCV20 plasmid in the E. coli DH5 transformants. PCR was performed in a 25 microliter reaction volume with 5 microliter of colony water as template. Colony PCR screening was successful.
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Easy to use protocol and less times on hand prepare PCR master mix and no need to use extra Mg++ ions in the reaction.
Nothing
GoTaq Hot Start PCR kit is ideal kit to be in the molecular biology lab especially colony PCR workstream.