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PCR question


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#1 Pazit

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Posted 06 March 2011 - 02:38 AM

what happens if after the first denaturation and annealing steps, I take the tube out of the PCR machine and leave it at room temperature for an hour or so? would the primers remain stuck to the template?
if not, how can I make sure the primers remain on the template for about an hour?

this is not a theoretical question, my experiment depends on the answer.

thanks.

#2 hobglobin

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Posted 06 March 2011 - 03:49 AM

yes, but also at sites where you don't want them. I.e. the temperature is low enough to allow unspecific binding and also the Taq still works, though only quite slow...Depending on the system you might get some extra bands or nothing happens, if you're lucky.
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#3 Pazit

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Posted 06 March 2011 - 04:38 AM

thanks! the experiment I'm doing is not exactly a PCR and doesn't involve a polymerase. I just need to get the primers to stick to the plasmid for about an hour at room temperature.

follow up question: what if after the one hour at room temperature I need to release the primers from the plasmid? I guess if I just heat it to 95 and cool it again, the primers will get back on the plasmid. but I don't want the primers anymore at that point. is there a way to do that?




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