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Antisense probe not being made

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#1 vetticus3



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Posted 31 March 2005 - 08:32 PM

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:



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