Nested PCR question - (Aug/16/2010 )

Hello again,

I am looking for some input on a nested PCR that I have planned.

We have been running multiplex PCR with 4 outer-gene loci, and then using that product to run RT-PCR's on respective inner genes. This has worked fine for our cell cDNA.

However, we are having problems with using this protocol on blood serum cDNA and looking at literature it proposed to just do the nested PCR by individually running the outer-gene primers and then running the inner primers individually.

Hopefully I am explaining myself right, so if anyone has any experience with this it is appreciated.

kindly see the attached site, that illustrates the principle of nested PCR.
nested PCR
in doing nested PCR, you have to do 2 PCR rounds, having the outcome of the first round used as a template for the second round.
hope ive helped !