Forward and reverse primers got very different Tm - what to do? (Dec/28/2009 )
Curtis on Jan 23 2010, 07:32 AM said:
Do you think this product would be suitable for cloning?
Probably -- it's tough to say without seeing the gel. Can you post a picture of it?
Is the smear you're seeing well separated from the 1 kb band or is it contiguous with it? How much amplification product did you get?
-HomeBrew-
sorry for the bad quality
forward primer CTTGGAATTCACCATGGACTCATCCAGGGCAATC
Tm=60.5
Reverse primer CTTGGTCGACTTTTTTGAAAGGGTTGTATTTAGC
Tm=55.7
and I put the annealing temperature at 51
I used 26 ng, 68 ng, and 1 ug of cDNA template but all give smear
-Curtis-