PCR GC rich genomic region

[jinglanl at stanford.edu]

Hi, everyone:

I am trying to clone a 250bp GC rich sequence from human genomic DNA by PCR,  primer F tm= 
60, primer R tm = 62, I us ethe following conmditon:

100ng human genomic DNA
2mM Mg
1x buffer
0.4uM each primer
1 M Betaine
0.125ul Promega Taq Pol

denature 97degree   30 sec
annealing 60 degree  30 sec
extension 72 degree  45sec

35 cycles

I got 2 single bands, the lower one is very strong and clear cut, looks like the specific product, 
but not at the right size; the upper one is faint, but at the right size.  I TA cloned both bands and 
sequenced, and it turned both bands are non-specific products, from two different but wrong 
chromosomes.

Any suggestion?

I also tried to do PCR after restriction enzyme digestion, but seemed no help.  Do I have to use 
some special Taq Pol for GC rich region?  Do I have to reduce 97 degree denature time to less 
than 10 seconds? 

Any suggestion?

Thanks a lot!

Jinglan Liu

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