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.
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.
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).
ps... it's FFRRRIIIIIIIIIIIDDDDAAAAYYYYYYY
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Antisense probe not being made
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