amplifying Genomic DNA

Jose de las Heras josenet at tiscali.co.uk
Mon Aug 7 20:32:24 EST 2006


"Rebecca Pickin" <RPickin at cvm.msstate.edu> wrote in message 
news:mailman.494.1154964172.20007.methods at net.bio.net...
> Hello all!
>
> I am trying to amplify the promoter region of my gene of interest to be
> able to clone it into a luciferase expression vector.  I have isolated
> genomic DNA from rat liver tissue (snap frozen, isolated with QIAGEN
> DNeasy kit, stored at 4oC for several weeks).  I am using gene specific
> primers at an appropriate annealing temperature (~5oC less than the
> melting temperature).  Unfortunately, even with increasing the MgCl2 and
> the primer concentration (from 1uM to 3uM) and decreasing the annealing
> temp, I'm not getting amplification of my 2kb band.  I did get a band
> about half the correct size.  I have not sequenced the bands so I dont'
> know what they are exactly.  Any suggestions as to ways to improve my PCR
> and get the bands I want???
>
> Thanks!
>
> Rebecca Roche Pickin, PhD
>
> Post Doctoral Associate
> Center For Environmental Health Sciences
> College of Veterinary Medicine - Basic Sciences
> Mail Stop 6100
> Mississippi State, MS 39762

Many promoter regions are very GC-righ. Take a look at your sequence. It may 
help to add some DMSO (5-7%) if that's the case.
I haven't encountered yet a promoter I wasn't able to amplify -eventually- 
and it mostly boiled down to using some DMSO, and perhaps increasing 
denaturing temperature. Sometimes it is necessary to increase the annealing 
temperature, because of other non-specific bands (it's as 'though the "real" 
template is hard to amplify, and other not-so-good matches amplify 
instead... but increasing the temperature and adding DMSO makes the primer 
annealing more specific). But in your case you only saw one band, so you may 
not need to do much. If you have a gradient PCR machine, I'd sstart by 
adding DMSO and trying several annealing temperatures with your "standard" 
MgCl2 and primer concentrations. But check the GC content of your 
sequence... it may not be a problem at all.

Jose 




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