SOS! asymmetric PCR, no PCR product - (Nov/24/2005 )

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Need your help!

Asymmetric PCR was used to amplify E. coli genomic sequences near the ends of Tn5 that inserted in the genome. I have run my PCR for about one weeks,but there is still no result. I use one primer that complementary to the sequence at an end of Tn5,another is a random primer.

the PcR reaction system:
genomic DNA 0.5ug
mgcl2 2.5mM
dNTP 0.2mM
Primer each 1mM
Takara Taq 0.5U
ddh2o to 100ul

cycles as following:
(1) 94' 15sec 58' 1min 72' 2min 5cycles
(2) 94' 15sec 30' 3min 72' 2min 1cycles
(3)94' 15sec 44' 3min 72' 2min 12cycles combinate with(1)

The late time ,i have fund primer band on the gel!

any suggestions for me ?

Thank you !!

hfrank

You can't expect to find a sharp band on a gel. You are getting amplification of random length fragments, so the best case you can expect is a smear. I think you should look at inverse PCR. Cut with an enzyme which cuts every 1KB or so in your genome (take account of the GC bias and methylation status... check that you can cut the DNA by running it and uncut genomic on a gel). The enzyme should not cut in the transposon. Dilute the DNA to encourage monomolecular reactions. Religate the DNA. PCR with primers directed outward from both ends of your TN5 insertion. This will create a PCR fragment containing portions of genomic DNA both upstream and downstream of your insertion site, linked at the RE cut site. I'm assuming you are doing this from a single colony clone which has an insertion at a single chromosomal site.

Thank you phage 434 for your timely help ! I'll have a try .I'm a novice to do PCR.Many thanks to you!