Do you have to PCR it, or could you cut a restriction fragment out of a
BAC prep and clone that instead?
Peter
On Wed, 3 Jun 2009, Christian Praetorius wrote:
> Hi,
> I have some trouble with the amplification of a human promoter, which
> shall be subsequently cloned to a luciferase expression vector.
> Initially I worked with genomic DNA, which was prepared in our lab and
> worked quite well for other applications. I finally also got the BAC
> clone containing the sequence in question.
> I tried doing PCR with different Mg-concentrations (from 1-4mM),
> different additives (betaine, DMSO, BSA), different annealing
> temperaturs as well as running a few cycles with a lower annealing
> temp. and then using a higher (and combinations of the methods above).
> The problem with this promoter is a stretch with 90-100% GC-content
> near to the transcription start site.
> I get either no product or a bunch of them with much lower sizes than
> expected, nested PCR gave the same results.
> Does anyone have any comments or ideas?
>> Christian
>> --
> X-no-Sig: yes
>
