i have a few Rhizobial isolates and i have to do 16s pcr with those followed by RE digestion for ARDRA analysis. I tried the pcr but i am not getting enough yield so as to give enough visualisable bands post RE digestion.
The genomic dna template that i am using has shearing. I run a PCR with those and i get a smear in the pcr too. sometimes above and sometimes below the specific band (1.5 kb) and sometimes at both the palces.
I tried boiling lysis rather than the entire genomic dna isolation, however i got nonspecific amplifications with it. (ok, how many microlitres of water do u resuspend the pellet in before putting it it the boiling?)
is it possible that the observed smearing in the pcr can be a result of the smear in the genimic dna ?