Hi,
I am having some problems with my PCR. I have performed a RNA extraction with RNeasy Mini Kit, and after that, DNase treatment (RQ1 RNase free Dnase, Promega) on 2ug of the RNA obtained from the extraction. Then I perfromed actin amplification to evaluate the yield of my experiments. I've loaded the products on a 2% agarose gel, and then I saw this:
in the second lane there's the pcr product obtained without DNase treatment,
the third lane is the pcr product after DNase treatment,
my question is: What is that smear? Can it be DNA?
It could be. Have the primers designed based on exon-exon junctions?
I think the cDNA concentration is high in your PCR reaction. Reduce the cDNA concentration and try amplifying, if you have standardized the amplification conditions like annealing temp. then you should get a crisp band.
but I don’t know why The same sample wituout Dnase treatment does not show The same smear.
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