well, i read the other post on pcr smearing and i thought i would throw in my own situation:
i am amplifying a couple of nuclear genes, with EF1-alpha being one of them, and have had pretty much no luck lately with either--lots of streaking.
i am using the following 50ul mix:
25uM MgCl2, 6ul
10x buffer, 5ul
dNTPs 10uM, 1ul
10uM primers, 2ul each
taq 0.3ul
DNA 3ul
my gDNA has been working out well for mitochondrial genes and i get clear bands with the above reaction, excepting that i only use 1ul of primer for mitochondrial genes.
i have had prior success with the two nuclear genes under the above conditions with vastly different annealing temps (60°C and 48°C), sometimes having to perform semi-nested PCR and now i have no luck. i have tried varying the amount of MgCl2 in the mix as well as using annealing temperatures in between that vast range and still i get smears. is this bacterial contamination? am i using an incorrect amount of something? not achieving bands now makes me wonder if something i'm using has gone bad....although my mitochondrial genes have always consistantly produced nice bands using the same taq, buffer...everything.
i'm pulling my hair out!
-ming
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