amplifying Genomic DNA

m.lumb at ucl.ac.uk m.lumb at ucl.ac.uk
Mon Aug 7 11:11:05 EST 2006


What enzyme are you using? I've had a lot of success with Phusion DNA
polymerase (from NEB) to pcr from genomic DNA. I have amplified up to
3.5 kb without any problem when both Taq and Pfu failed. Might be worth
a try. No affiliation just a happy customer.

Mike Lumb

University college London
UK

Rebecca Pickin wrote:
> 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



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