a smeared Gen-DNA template ---> smear and less yield on pcr ? - (Aug/06/2013 )
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 ?
You are likely using far too many cells in your PCR, or far too much template in a PCR reaction. Touch a tip to a colony and mix in 50 ul of water. Vortex and use 1 ul as a PCR template. Extend the initial 96 denaturing time to 4 minutes in your PCR. If you prep DNA, dilute it by 100x or 1000x before adding it to your PCR reaction. You should see only a single band on a good PCR product.