how to amplify viral-vased shRNA - (Feb/20/2008 )

Hi, dear all:
I am going to receive a viral-vector based shRNA. since i am not familliar with this kind of stuff, can any1 tell me how to amplify it? just like common plasmid DNA and transform it into E.Coli compent cells?
THANKS..

-Niu-

If you're going to reveive vector, you might want to make sure what the vector exactly contains. If it e.g. contains LTR's, you probably want to grow you competent cells @ 30°C (and 180 rpm) at all steps instead of the standard (37°C) due to instability of LTR's in e. coli. If growing @ 30°C, take a little longer than normal (e.g. 90 minutes in SOC) and you should be fine.

-vairus-

Or try to transform your DNA into recombinase - cell line, such as STBL3. Otherwise, your plasmid will be screwed.

-Madrius-

As suggested, try recombination negative cells (Stbl3 or SURE) and also try to sequence it after growing to confirm the presence of shRNA and its sequence.

-scolix-

thanks , guys.
Niu

QUOTE (scolix @ Feb 21 2008, 07:46 AM)

As suggested, try recombination negative cells (Stbl3 or SURE) and also try to sequence it after growing to confirm the presence of shRNA and its sequence.

-Niu-