Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum
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I am doing a rt-pcr quantification assay using sybr green. I am trying to obtain my standard curve with plasmid DNA contaiming my gene of interest. Does it matter if the plasmid is linear or not?
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- Joined: Oct 29 2004 11:07 am
- Location: UC Davis
You must linearize your plasmid before attempting qPCR. circular or supercoiled plasmid will not amplify with the same efficiency as dsDNA. In many cases, your primer annealing site may be obscured due to twisting of the plasmid. Taq pol will also have a difficult time getting itself to latch onto a circular piece of DNA.
While uncut plasmid is a perfectly acceptable target for certain applications, i.e. colony PCR, the extreme sensitivity of qPCR requires you linearize the plasmid
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- Joined: Jan 03 2003 3:45 pm
- Location: New York University
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