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Antisense probe not being made


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#1 vetticus3

vetticus3

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Posted 31 March 2005 - 08:32 PM

Hello,
I have a problem with one of my probes.  
I was given a plasmid containing the insert i want.  It's my insert with a pGemT easy vector.  I digested it with Nae1 (aka Pdi1), but when i ran the gel out there was a *faint* band above (about 1kb) the cut plamid and (about 4 kb)) above the uncut plasmid.
Totally ignoring this, i went ahead and invitro transcibed it, using T7 maxiscript.  Ran it out on a denaturing gel, and nothing. :(
Did it again... nothing.  :huh:
Figured, ah that the plasmid wasn't cut... recut (other band so faint but there).... nothing happened.   :(
Figured other band was the thorn in my side, gel purified... invitro transcribed, and nothing again. :angry:
The only thing i can think of is that instead of T7, it should be a Sp6 RNA polymerase.  but this is totally against the instructions i've been given. :)
Is there anything else that could explain this (all other transcription worked fine, and they were done at the same time). :huh:

Thanks,
Vetticus

ps...  it's FFRRRIIIIIIIIIIIDDDDAAAAYYYYYYY B)




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