problem with smear - (Dec/02/2006 )
i'm trying to amplify the 5' UTR of a gene using 5' RACE protocol ...but so far i've bn unable to get the specific amplification...large amount of non specific amplification has been observed ...and hence a smear is seen everytime the products are electrophoresed on 2% agarose.
i've been using the following steps:
denaturation- 94 degree celcius -3'
annealing- 32 degree celcius -2'
extension- 72 degree celcius -15'
followed by 30 cycles of:
denaturation- 94 degree celcius -30 sec
annealing- 60 degree celcius -30 sec
extension- 72 degree celcius -2'
the primers used are--oligo dT anchor primer(Tm-77.1) and gene specific Reverse primer(Tm 69.3)
The cDNA has been screened for the verification of polyA tailing and is found viable.
am wondering what modifications could help me out with the problem.
check ur template, a higher conc also gives a smear in ur pcr rxn. also check ur mgcl2 conc in the rxn.