(by prae from gmx.net)
Wed Jun 3 09:27:49 EST 2009
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?
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