Hello everyone! I need to amplify a specific DNA fragment in order to do Sanger sequencing for the detection of a point mutation in human patients. The fragment is in an exon of my gene of interest. I designed specific primers of 20nt each, not annealing with other genes (verified by BLAST), both with the same Tm, 50% of GC and 50% of AT. I performed PCR with Phusion High Fidelity polymerase, adding firstly DMSO 5%, then Betaine 1M, and increasing Tm, but I always obtained a lighter "aspecific" band of 1500bp along with the specific PCR product of 500bp. I then decided to design new primers, but same thing happened. In electrophoresis, I obtained one band of 370bp and the same lighter band at 1500bp. Negative control is totally clean, so I don't have contaminants in my solutions. I run PCR on DNA samples from 10 different individuals and observed the same 1500bp aspecific band for all of them. I couldn't find any pseudogene or mtDNA alignement. I used RNAse when I extraxted DNA from blood. Could anyone tell me where the problem might be? I attached the picture of the electophoresis run. Thank you very much!
Since its only one band and very faint, i would reduce the number of cycles
Don't forget that you can bypass the 1500 band if you purify the other one
You can try to increase the annealing temperature (even more) and reduce extension time in PCR (to avoid the long product). You can also excise the band of interest from the gel to sequence. Try also make different combinations with the different primers. you may find a combination that does not result in that unspecific amplification.
You could try increasing the annealing temperature to try to get rid of the larger band and even try a "pcr" with only one primer to see if either primer generates this band non specifically. If you really want to know what it is the purify the band and sequence it
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