degenerate primer - optimizing degenerate primers (Feb/22/2003 )
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.
You could improve your chance by designing primers based on multiple alignments of a family of protein or nucleotide sequences. For more info, see