I am running HPV PCR with the product size of 450bp. the products are coming at 500 and 464 respectively. also when I run the PCR again with the same conditions, there are no bands visible. I have tried new working solution of primers, new gradient temperatures and new extension cycle. only smearing is visible with no bands. on nanodrop the purity for DNA is 1.8 with good concentrations respectively. is my DNA degraded or something. how should I proceed with this.
When you say you are running the pcr again do you mean re amplifying pcr product or just running fresh dna samples?
What does your no template control look like. It sound like you have over amplification due to pcr contamination from previous runs. You should run a couple of water samples .these will give a product if you have any contamination and over amplificaion will cause the pcr product to self anneal and cause a smear
Paul Rutland I am running PCR again by fresh dna samples. and still getting smears. i did ran a NTC, it also had smear. but the positive control amplified nicely.
Do you have a picture of that gel please Syeda? Over amplification smears and single primer binding smears are often smaller than dna degradation smears. Amplification in a NTC always means contamination to me but the positive control amplifies cleanly which is not typical of contamination. How much total dna are you amplifying in both your normal samples and positive control? It would be interesting to see what one dna sample (not amplified) looks like on agarose gel and also a mixed sample to check if your dna samples have pcr inhibitors. What I mean is run positive control also run one of your samples, also run a sample of positive control and your sample in the same tube to see if your sample spoils the amplification of the positive control as well as the ntc sample.
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