Time course for siRNA inhibition - When is the peak of inhibition after transfection ? (Jun/26/2006 )
QUOTE (genehunter-1 @ Jul 1 2006, 12:25 AM)
QUOTE (dnafactory @ Jun 27 2006, 08:09 AM)
QUOTE (stardust @ Jun 27 2006, 05:01 PM)
Hi there!
May I ask which sequence you used to target EGFP? Mine doesn't work as well as yours
Thanks, Stardust
We bought one from Ambion to be used as a control siRNA instead of scrambled ones and if we try to transfect both DNA and siRNA at the same time, it inhibits the transfection (or expression, we don't know) of the cDNAs
dnafactory, Just FYI:
I checked co-transfection of this siRNA with pCMVb-gal and found no inhibition on the expression of beta-gal at siRNA dosages that gave ~90% inhibition of its target sequence EGFP.
Did you buy the predesigned that they give you without sequence info? We tried that one and gave all of us in the lab the same problem. But if it worked for you, maybe we should give it a try again. We transfected U2OS with Lip2000, in a 35mm dish with 60pmol siRNA and 1,2ug DNA. Thanks in advance for your reply
-dnafactory-
QUOTE (dnafactory @ Jun 30 2006, 04:31 PM)
QUOTE (genehunter-1 @ Jul 1 2006, 12:25 AM)
QUOTE (dnafactory @ Jun 27 2006, 08:09 AM)
QUOTE (stardust @ Jun 27 2006, 05:01 PM)
Hi there!
May I ask which sequence you used to target EGFP? Mine doesn't work as well as yours
Thanks, Stardust
We bought one from Ambion to be used as a control siRNA instead of scrambled ones and if we try to transfect both DNA and siRNA at the same time, it inhibits the transfection (or expression, we don't know) of the cDNAs
dnafactory, Just FYI:
I checked co-transfection of this siRNA with pCMVb-gal and found no inhibition on the expression of beta-gal at siRNA dosages that gave ~90% inhibition of its target sequence EGFP.
Did you buy the predesigned that they give you without sequence info? We tried that one and gave all of us in the lab the same problem. But if it worked for you, maybe we should give it a try again. We transfected U2OS with Lip2000, in a 35mm dish with 60pmol siRNA and 1,2ug DNA. Thanks in advance for your reply
It is pre-designed. I have sequences info came along with the product (27mer dicer substrate, IDT ReadyMade part #: 23467750 EGFP-S1 DS Positive Control). For 293-EGFPluc cells in 96 well plates I used 0.5-2.5 ul of 1 uM solution of siRNA, and 0.2 ug pCMVb-gal per well in 100 ul.
-genehunter-1-