getting smear in PCR - (Dec/28/2006 )
i am doing pcr more than a couple of weeks,i am using plant DNA after running PCR i am getting smear,but DNA is getting amplifying but no bands i tried in altering everything in the mix still its like that please helpme its urgent
Smearing in PCR often results from too much template or too much polymerase. As a first test, try doing a serial dilution of your template (1:10, 1:100, 1:1000). Also try using less polymerase. If the smearing remains, we'll try to come up with other suggestions.
Smear could be cause by: to much template and primers, degrate DNA, to much polimerase or to much MgCL2.check each one( but not at the same time, because you will not know the real cause of the problem).I will recommend that begin with the template check the concentration of the stock and mantein a good ratio template/primer. The polimerase should be no more than 5U/50microL per rx and check that you're adding the correct M of MgCl2. If all of this fail and still seen smear and no band then this means that you DNA is degrate.
Are these known-working primers? Try raising the annealing temperature (and I agree that serial dilutions of the temlate is also an excellent first step). Try annealing at 55, 60, 65, or use a gradient machine.