I have designed primers that bind to conserved regions,so that variable regions inbetween the conserved regions get amplified.
My main aim is to detect the microbial diversity in a given sample by amplifying the variable regions of different microbes.
I used to amplify variable regions and get a 340 bp product and with this product i used to clone it by topo vector and later on used to sequence each single clone by sanger sequencing and the sequences were very fine.
But when i gave the 340 bp PCR product directly for sanger sequencing there was presence of muliple peaks.so iam stuck here.Why my sequencing is not working.what is the problem? i give exosap treatment before sequencing.some one please help me out what went wrong in my experiment or is it not possible to get sequences of variable region amplicon?
please help me out with your valuable suggestion