Promoter amplification

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Fri Jun 5 15:48:48 EST 2009


In <78pr1fF1krs6cU1 from mid.individual.net>,
 Christian Praetorius <prae from gmx.net> wrote:

> Peter Ellis <pjie2 from cam.ac.uk> wrote:
>
>>Do you have to PCR it, 
>
> No, I don't have to. Its simply easier, when it works.
>
>>or could you cut a restriction fragment out of a 
>>BAC prep and clone that instead?
>
> Good sugestion. I am actually digging into it, and it seems possible.
> But I have to change the MCS of my plasmid but inserting a oligo with
> the right sites. 

I face a very similar situation.  I have a promoter/enhancer that is
approximately 10 kb in size.  The fragment I have begins approximately
1 kb before the farthest known protein binding site, where expression
of the protein increases promoter activity rather dramatically (about 
30-fold).  The fragment ends about 100 bp after the known transcription
start-site.

We have accidentally discovered that there is an additional 600 bp
_AFTER_ the transcription start-site that also increase promoter
activity, most likely by participating in enhanceosome formation.

There were no convenient restriction sites to use, and PCR of this
fragment failed time & again.

So, we ended up synthesizing the fragment as a "mini-gene"; we used
a German company called Mr. Gene, but lots of places offer very
competitive prices now.

Because adding this fragment added a long 5'UTR, not to mention
part of an ORF, we modified the reporter to include the EMCV IRES
before the luciferase ORF.  We decided to include the EMCV IRES
as part of the "mini-gene" that was synthesized.  

The total length was about 1200 bp, and it cost us $600.00 + 
$50 in shipping charges.  Synthesis took less than less than 4
weeks.

I don't know how long your piece is, but you could consider having
it synthesized as a "mini-gene".

Ashok

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