getting rid of RCR smears - (Apr/17/2007 )
Hi!
lately I've started having smears in addition to the desired band. the smear is faint in the negative control(without DNA) and gets stronger in the other samples. Ive tried increasing anealing temprature and changing primer concentration. I also tried adding DMSO with no luck. using new primer stocks did not help.
Please help!
have you tried to play around with the concentration of MgCl2? Or perhaps you have too much DNA template and end up smearing?
So I guess you have your desired bands but just smearing at the background? It might be DNA degradation. Perhaps coming from impurity of the DNA template.
So I guess you have your desired bands but just smearing at the background? It might be DNA degradation. Perhaps coming from impurity of the DNA template.
changes in MgCl2 didnt help, and the problem repeats itself when using different templates , so I dont think it has anything to do with the template. I use very low template concentrations , when I use smaller template concentrations its harder for me to see the band inside the smear
could try reducing the number of cycles. The product yield of a PCR reaction plateaus after about 30 cycles. So if you drop the number of cycles, the band of interest will remain strong but bands from side product will be reduced.