BioTechniques Molecular Biology Forum brings bench scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the DNA and General PCR Methods forum.
Q: I'm trying to do Hot Start PCR using single buccal cell as my sample and planning to put the cell directly into the PCR reagent mix. Will this work? Recommended initial activation time is 95 degrees C for five minutes for Qiagen's HotStar PCR kit. Should I increase the time to 10-15 minutes for the initial denaturation?
A: For genomic PCR, adding the cell directly to the PCR mix should work. Initial denaturation for 5 minutes should be OK. Longer denaturation may shorten the life of the polymerase. If you are doing qRT-PCR, try Ambion Single Cell-to-CT Kit.
A: If it's single cell genomic, that means you're aiming to amplify only one or two copies of the target gene, which is challenging...
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