Cloning into shRNA vectors - (Jan/18/2005 )

How do people get on cloning oligos for shRNA expression into shRNA expression vectors? Any tips or tricks? I've just been cloning a couple and it was very difficult!"

David
Expresson
http://www.expresson.com

-expresson_help-

Just synthesize the oligos(sense and antisense) with two overhand ends as restriction enzyme site and anneal the two oligos. Treat it as common insert to clone under polIII promoter.

Hopes this help

cheers

-biotin-

hi
for improving efficency of cloning, and especially if you have compatible ends, you should order phosphorylated oligos.

-fred_33-

QUOTE (expresson_help @ Jan 18 2005, 03:54 PM)

Hi,

Check invitrogen's pENTR/U6 or pENTR/H1/TO vector. A simple 5 min cloning.

Cheers

Knight

-Knight-

Has anyone had any success using Invitrogen's pENTR/H1TO shRNA system? Cloning and construction of the vectors was easy, and now I have several stable lines (...like 50 for various targets). The problem I have now is detecting a knockdown. My control shRNA vector dosen't seem to knock down its target, even though the sequence has been validated and published. I have tried to induce expression in Helas using both tetracycline and doxycycline, with no success. Any advice??

-janam-