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Forward and reverse primers got very different Tm - what to do? (Dec/28/2009 )

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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
Attached Image

-Curtis-
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