I extracted genomic DNA from two different cell types (HEK293T and HDFs) and performed PCR reaction by using a same primer set. The PCR is positive with HEK293T template but not with that from HDF. What could be the possible reason? I tried it multiple times but results are consistent. The expected amplicon size is 3.0kb.
Hi Nasir Javaid , Did you check the quality of DNA isolated from HDF? Sounds like there is a problem with the quality of DNA. Try running a 0.8% or 1% agarose gel and check the quality of DNA or trying to perform any one of the housekeeping gene PCR and check on an agarose gel.
Ashis Kumar I did PCR by using a different set of primer and it was working on both kind of templates. I wonder could it be because of the different genomic complexity (secondary structures etc.) in both cell types which is hindering the binding of primers in HDF but not in HEK293T?
Possibly the sample that does not amplify has a polymorphism under the 3' end of one of the primers. If the 3' end of a pcr primer does not bind then Taq polymerase cannot extend and there will be no pcr product. Try lowering the annealing temperature 6C and if this works then you probably have a mismatch under one primer in that cell line
Paul Rutland I reduced the annealing temperature to 5C but it didn't work. Is there any other possibility
An insertion between the primers or a deletion of one primer sequence in the non working template or a rearrangement of the amplified sequence so that the primers now face away from each other not towards each other would all cause the pcr to fail.
It may be as you suggest that the sequence is long 3kb and complex or high GC in which case try amplifying with a gradient of dmso in steps of dmso concentration of 1% 2%......8% and possibly do the pcr in the presence of 1Molar final concentration of betaine to sort out template secondary structure problems like hairpin looping or G quadruplex and straighten out the template/amplimer.
Try also using less template dna in case of pcr inhibitors....sometimes diluting the inhibitor has a bigger positive effect than the negative effect of using less template
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