Antisense probe not being made - (Mar/31/2005 )
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
OK, my 2 cents.
pGEM-T Easy accepts PCR products non-directionally. I hope that the people that gave you the plasmid did a test digest to determine the directionality of the insert. As you know, the T7 and SP6 promoters flank the insert site and point in opposite directions.
What does Nae I do? linearize? Where does it cut?
Nae1, according to the people who sent me the plasmid and the instructions for use, should linearise the plasmid. According to the promega map, it cuts in the f1 ori site.
I'm assuming they've done the direction test, but well, getting the info on this like getting blood from stone.
Any hints on what else to try?
i think maybe you should try and get the plasmid sequenced...that should give you some info. it almost sounds like it's contaminated (because of those extra non-digested bands). if it is contaminated you may need to transform some bacteria and then grow up plasmid from a single colony to make sure its ok.
so what are you making the probes for, and what kind of probes are they? because if your insert isnt HUGE, you can always PCR the insert out (using primers that anneal outside of the T7 and Sp6 sites so that they are included in your PCR product), then use the PCR product for your transcription reaction. I do this to make T7 and Sp6 RNA probes for in situ hybridisations and it works really well...
nuclear run on transcription assay.
Thanks for the PCR method. will help alot!
I think i found out what was going wrong. bad handwriting!!! the pGem T plasmid.... is probably pGem 4Z..... and Nae1 is more likely Nde1.... ahhhhh, all is well in the world, i will not cuss or cry today
sometimes, the most dumbfounding problems are caused by the silliest things.
don't we all have similar stories?