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