cloning: band at different site than desired after RE digestion - (Jan/04/2013 )
shivu11 on Mon Jan 7 09:11:27 2013 said:
I do not estimate the dna conc. We usually isolate plasmid from 5ml culture, inoculated overnight. Gel shows gud intensity band. and my plasmid size is 5000bp. and band which I am getting is at 3000-2500bp.
Well, you will likely have a bucket load of DNA from a 5ml mini prep, so using that as neat template for PCR may be your problem. Have you considered trying the dilutions like I suggested?
Are you open to the possibility that this 2500-3000bp band you are seeing is uncut plasmid? Or have you dismissed that idea, or ruled it out someway or other?
thanks for the reply. I am trying to clone the gene again, and hope to get the success this time, Thanks again to all for ur time and patience.