Protocol Online logo
Top : Forum Archives: : Molecular Biology

Degenerate primers for nested PCR? - (Nov/20/2005 )

I'm trying to amplify fly homeobox sequences using degenerate primers.
Degenerate primers are designed after reference nucleotide and amino acids sequences of fruit flies, beetles and honeybees.
The PCR product showed several bands on agarose gels.
I selected one possible band and cloned it.
But it was not the target gene, but an another gene.
I think there are many different nucleotide sequences in a band.
My opinion is to design another degenerate internal primers for nested PCR.
But my professor thinks that nested PCR with degenerate internal primers will provide nonspecific bands only and so it's useless.
I don't know what to do now.
Should I persuade my professor? unsure.gif


You will always get a lot of "noise" with degenerate PCR. You will have to make a mini library with PCR products and sequence. It is also a good idea to make lots of different primers as you might find that one set work while another don't.


Software for sequencing DNA

-Daniel Tillett-