nested PCR: purification? - (Jun/29/2006 )
is it necessary to purify 1st round PCR products before going on to the second round?
i've been trying to amplify some cDNA made from viral RNA and have been having some difficulty. i've had one successful amplification but all my others seem to be producing bands not at all near my target size and i was just curious if purification between 1st and 2nd round is necessary or even helpful.
We never do it. We do RT-PCR on viral RNA as well, also with nested PCR. First thing to do is (if possible) optimise your PCR (both inner and outer) on DNA, before switching to RNA. Just try to make sure your nested procedure works, and that you can repeat your results.
Did you perfrom your "other" amplifications from the same starting material (meaning: have you had your RNA "freeze-thawed" often)?
The way we go is: we do outer cDNA synthesis, add 5 µl of a 20 µl reaction to the outer PCR (we correct for dNTP, antisense primer and Mg, we PCR for 40 cycles) in a 50 µl reaction. Of this, we just add up to 5 µl to an inner PCR reaction (I mostly just use one which workes fine, some others here use up to 5 µl, 30-35 cycles). About one year ago, we started working with "one step RT-PCR" for our outer PCR, and changed nothing on the inner PCR and it's still working fine.
The reasons for doing nested PCR is to get more specific product and also to get greater yields than with single PCR. A purification step after the outer PCR wont' help much I think, you'll lose time and maybe even some DNA (after the outer, there's mostly not too much to see on gel anyway, and no purification method results in a 100% recovery).
Hope this helps.