I want to amplify the human beta globin 3' UTR from Human genomic DNA. I have done gradient PCR. But could not get my desired product. The Tm of Forward primer is 66 Celsius and Reverse Primer is 61 Celsius. I amplified GAPDH from DNA but 3UTR is not amplified. After PCR I have run gel electrophoresis which indicates only primers dimers.
Hi Ali
a difference of Tm more than 2°c is too high to allow perfect hybridation of the primers at same time/point. try to fix that point. secondly, I suggest you to test your primers in silico (http://genome.ucsc.edu/cgi-bin/hgPcr) just to be sure you're the right target.
stay safe
fred
Hi Ali,
I agree with Frederic that it is necessary to check if your primers are unique to your sequence, I always use NCBI BLAST for PCR primer to see if it matches other sequences (in human genome in your case).
There are many parameters you could try to change.
I would try to design another primer pair.
For the primers you have, if you can:
Change elongation time for PCR reaction,
Increase number of cycles to 40,
Check how much DNA you use in PCR reaction (20-100ng should work in 20 ul reaction),
Increase PCR reaction volume to 50ul,
Change concentration of salt in PCR reaction,
Change PCR polymerase,
If the target sequence is GC-rich, modify PCR for GC-rich sequences (I don't remember, please google it),
Try nested PCR.
How long is the sequence you need to amplify?
Thanks, Fredric and Maria
I have done primer blast my primers have matched with desired sequence (HBB-3'UTR= 134 BP). I have also done in slico PCR which give me exact sequence of this gene.
In a lab, I tried 5 different annealing temperatures (52,54,56,58,60). But nothing found ( even single band) found in Gel.
redesigning primers with no more than 2°c Tm difference shoulb be better.
or do touchdown PCR to allow hybridyzation of both primers during the cycles.
all the best
fred
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