PCR question

last updated by

JackBean 13 years, 8 months ago

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- March 6, 2011 at 10:35 am #14599
  unibas
  Participant
  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.
- March 6, 2011 at 11:40 am #103755
  DRT23
  Participant
  Since lower temprature makes DNA-primer binding more stabile and while annealing temprature is about 50 C and room temprature is 25 C, the primers should remain stuck to the template. But, if you want to be sure, you may compare this procedure (removing the tube after the first annealing step and keeping in room temprature for an hour) with the regular procedure by using a quantitative-PCR. Starting with 2 tubes that are identical in terms of amounts and types of ingredients, if primers remains stuck to templates in your procedure, there will be same amount of DNA products at the end of amplification, after same number of cycles for both procedures.
- March 6, 2011 at 12:35 pm #103757
  unibas
  Participant
  thanks! I’ll do that.
  follow up question: suppose the primers remain bound after one hour at room temperature. after this one hour, is it possible 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.
- March 7, 2011 at 12:57 pm #103785
  JackBean
  Participant
  you could increase the ionic strength or cool it slowly, than it will take enough time and rather longer chains should anneal.
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