plasmid or linearized plasmid??? Does it matter?

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plasmid or linearized plasmid??? Does it matter?

Postby khowes » Jan 10 2005 6:21 pm

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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Postby ParasiteGuy » Jan 18 2005 4:33 pm

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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