I have extracted viral RNA as well Proviral.Viral RNA is further converted to cDNA.Am using the same primer for amplification of cDNA as well proviral.but unfortunately,am getting smear in both cases.Smears of both low molecular weight as well high mol.weight.
what can be the posible reason of these smear
smears in pcr often mean one of 2 things. Either only one primer is annealing so you get random length extension off one primer or you have post pcr contamination of one of your reagents and you are getting over amplification where the amplimer anneals to itself and extends in random fashion. If this is the case then the negative control ( no dna template in the tube) will show an amplification smear as well as the samples. Is this happening in your negative control ?
Dear Paul I didnt run the negative control the way you mentioned.now i will try this.
Dear Laurence I try that but i get stuck when i get smears even using proviral DNA.
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