Hello all.
I have used consensus PG/MY primers for HPV typing and the PCR was standardized with HPV positive control. But suddenly I am getting smears instead of bands though I used same conditions as well as same preparation steps. I tried the PCR several times by changing concentration of primers, Mgcl2 as well as samples but still getting the same result. I am attaching the picture of the gel. Please help me with your suggestions.
I think that you have post pcr contamination of one of your reagents or working area. A tiny amount of contamination amplifies really well and eventually there is too much and it anneals to itself into long concatamers ( smear). If you run a no sample control and get the same smear when you should have no amplification then you have contamination. It is hard to get rid of and the quickest way is to redesign the primers so that one primer is outside the current amplimer primers
Hi,
I agree with Dr. Rutland above. There seems to be some amplicon (may be from comtamination) which is in good amount and yielsd such type of smear. It is sstill not clear to me, if you are doing the same PCR for typing of strains or trying to do a re-PCR amplification of a previously purified amplicon. In the later case, you need to take a very small of purified amplicon for a re-PCR. See if you have changed some reagent recently
The PCR itself could have run well however, try increasing the concentration of the gel or reducing the voltage during electrophoresis. Higher voltages cause the amplicons to run much faster without resolving into bands and instead producing smears.
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