In some of my nested PCR experiments, I noted the appearance of bands referring to both first- and second-round products. Is there a relation with the quantity of the first-round product that I used as a template for my second-round reactions? When I used a reduced quantity of the template, the bands of the first-round disappeared. Could anyone possibly give me an explanation of what was happening?
Thanks a lot in advance!
Dear Rashad,
It looks like you have a very efficient amplification at the first round and when you take some sample to the second round reaction and analyze your gel you still have a sufficient amount to see the first round band. So you basically answered your question when reduced the amount of template for the second round and could not see the first round band.
Good luck,
Alexey
Dear Rashad,
Generally when you do a nested PCR by taking an aliquot of the first round PCR product, you should not see your First round PCR product in the gel. Since you are using an internal primer, it will make the PCR more specific in the second round. If your primer pair is specific, you shouldn't see your First round PCR product. But as you yourself have suggested when you dilute it, you don't see it. Because when you are diluting it, there will be very few copies of the First round PCR product. So, now you see a better amplification. Hope that answers your question.
Sreemanta Pramanik
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