Gateway cloning and expression

Jayakumar, R R.Jayakumar at roswellpark.org
Thu Jan 12 17:02:09 EST 2006


Did you try screening clones for position integration effect?  I am not sure how this vectors integrate.  But generally genomic integration is random and it is necessary that they integrate into a transcriptionally active loci in the genome so as to get stable transfection.  This may necessitate cloning and screening at least 20 - 30 clones for stable transfection by RT-PCR or westerns (better).
jai

-----Original Message-----
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu]On Behalf Of Simon Dawson
Sent: Thursday, January 12, 2006 9:13 AM
To: methods at magpie.bio.indiana.edu
Subject: Gateway cloning and expression


Hi,
	Does anyone out there have experience with the Gateway cloning system 
of Invitrogen? Anyone tried using there vectors pcDNA-DEST40 and 
pcDNA-DEST47?
	I've tried any number of cDNAs in them and get no expression on 
transfection into mammalian cells. Sequenced them and they're all OK. 
I'd be interested to hear what experiences anyone else out there has had 
with the GateWay system.
	Cheers,


		Simon.
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