I am trying to get my degenerate primers to work. Their Tms range from 40 to 70C, I have used many touchdowns from 65 to 38 but they don't seem to work. I decrease the anneling temp. by 0.5C in touchdown, does it need to be more fine? I have tried RT-PCR as well as PCR using gDNA or cDNA.I use qiagen's hotstart/ 1 step RT kit, which had worked for all other primers, so I believe Mg concn. or dNTPs may not be a problem.I'll appreciate any suggestions.
Also, can u think of any other method to get sequence of an unknown gene in less studied organism, except doing amino acid homology and designing degenerate primers.
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