Hi tomato, I tried to analyze your primer (P7-43DF3/R1 ), it seems that your primers have high primer dimers...
CACGGGATATG TT RT TG ATAAG CATGT
____________C CA CT AT TTCAA GGACACCATTTCTG
As suggested by lab rat and others, 5% of the DMSO can be substitute your BSA.
Also, try run a gradient temp PCR range from 50 to 65.
I suspect your annealing temperature should be around 56~58C5 (althought published temperature might be different).
Here are my humble suggestion:
Try to Keep your MgCl2 final conc at 1.8 or 2.0, DMSO 5%(I use in all my pcr, works fine)
Also, I doubt whether your template got starch contamination... as the smearing is quite heavy.
Furthermore, For Takara Taq 10X buffer there are 2 types, One is not mgcl2 free and another is mgcl2, which could be the problem. Hope you didn't took the wrong one...
just my 2 cents.
Edited by adrian kohsf, 07 December 2009 - 03:39 PM.
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