Nested primers - (Jun/15/2008 )

Hi

I tried amplifying my genes with a set of primers but it did not work. I was told to use nested primers.
('''' gene, ----- 1st set of primers, ***nested primers) as shown below.

5' '''''''''''''''(''''''''''''''''''''''ORF''''''''''''''''''''''''''''')''''''''''''''''''3'
----- ------
**** ****

Was wondering how these nested primers could help. Can someone explain?

Also, any suggestions on how i could troubleshoot the amplification of my genes (besides nested primers) would greatly be appreciated.
Thanks in advance:)

I don't know how accurately your picture describes the primer positions. For them to be nested, the 3' end of second (inner) pair must be inside the region amplified by he outer first pair. The idea is that the first (outer) pair amplifies the DNA in a (perhaps) non-specific manner, producing a mix of products. The second (inner) pair of primers uses the first PCR result as a template to amplify only the desired target. This is a good strategy if your first set of primers are not selective enough.

oops...my diagram's position went off. they are supposed to look like this.thanks!

5' '''''''''''''''(''''''''''''''''''''''ORF''''''''''''''''''''''''''''')''''''''''''''''''3'
----- ------
**** ****

Im trying to amplify th ORF region only and start after the methionine. so does that mean that
my second set of nested primers must be designed at the codon after the MET. thanks

the diagram is still not displayed right. anyway the first set should be around 5'end and 3'end(outside ORF) then
the nested primers overlapped the first set of primers into the ORF region. sorry bout that.

u didnt gave any information about ur template or primers. Is ur template is cDNA or genomic DNA are u using genespecific primers or degenerate.

If your using cDNA, the trancript levels of ur gene in the source, its GC content, your oligo GC content all matters for a successful PCR.

cheers
Gene tag