We did some nested pcr in our facility, without purifying anything, the bands appeared thick, some non specific bands showed up too

Maybe u had little dna to begin with that resulted in a lower amount

U can try to repeat the first run to increase the primary band then perform nested on that

Note that the concentration will not reflect on the sample

And when purifying a pcr product, its advisable to load the whole volume rather than the usual 3 or 5 ml

Purification may not be needed if your DNA was extracted with high purity. You can proceed to secondary PCR. We have done this successfully without purification, yet with excellent outcome.

Hey Andrew,

from our experience nested PCRs can be done without purification.

When we do a routine optimized nested PCR for one of our lab courses we omit the clean-up. We use a significantly lower concentration of primers in the first reaction though, so they get completely used up in the first reaction and can't pose any problems in the second one.

When I run quick nested PCR which I do not bother optimizing, I clean-up with ExoSAP only. You don't get the proper purification of column-based kits but It's quick, it gets rid of the primers and you don't have to worry about the loss of template.

THANK YOU for the replies. Your input has given me something new to try. I will let you all know how it goes. Thanks!

Andrew