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How to amplify very short PCR template


Best Answer phage434, 05 August 2013 - 01:34 PM

You need to dilute your first PCR reaction a LOT. Like 100x to 1000x. You may need to add more primer than you are used to. Primer will be a major part of your final product -- unlike longer PCR reactions.  Try increasing primer by 5x - 10x and highly diluting your previous PCR template. You may need to up Mg++ concentration to balance the increased primer amounts.

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#1 noagilat

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Posted 04 August 2013 - 11:36 PM

I used PCR on a 70 bp DNA template (bp, not Kb), and got no results at all (I used Gel electrophoresis and did not see the 70 bp DNA).

I should mention that the 70 bp template I used was a former PCR result I got from a lambda template of 48 Kb.

I'll be happy to receive any advice in this matter.

Thank you.



#2 doxorubicin

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Posted 05 August 2013 - 05:07 AM

That size is hard to see with Gel Electrophoresis, but would be a great size for qPCR.  Do you need to see the band or do you just need to know the product was made? 



#3 pcrman

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Posted 05 August 2013 - 01:06 PM

70 bp templates are still amplifiable and a product of 70 bp can be resolved and seen on a 2% agarose gel. If you did not see the band, you need to troubleshoot your PCR reaction. Have you diluted the template from the first PCR? Stuff directly from the first PCR reaction without dilution may inhibit the 2nd reaction.

#4 phage434

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Posted 05 August 2013 - 01:34 PM   Best Answer

You need to dilute your first PCR reaction a LOT. Like 100x to 1000x. You may need to add more primer than you are used to. Primer will be a major part of your final product -- unlike longer PCR reactions.  Try increasing primer by 5x - 10x and highly diluting your previous PCR template. You may need to up Mg++ concentration to balance the increased primer amounts.



#5 criscastells

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Posted 21 August 2013 - 03:25 AM

I have amplified fragments of 80 bp and have had no problems to visualize them with agarose gel. Anyway I used a higher concentration (4%). I would try to modify PCR parameters because it is possible that the fragment has not been amplified. A positive control may be really useful.






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