supF selection, pcDNAI-neo

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Tue Nov 12 19:11:58 EST 1996


On 12 Nov 1996 16:53:25 -0500, Ian A. York <iayork at panix.com> wrote:
>I've made a reasonable number of pcDNA1Neo plasmids, as well as other SupF
>plasmids (pCDM8, pcDNA1) and have not had the trouble you describe, so
>it's probably not intrinsic to the plasmid.  I grow the plasmids in
>MC1061/P3 and select with Amp 30 ug/ml + Tet 7.5 ug/ml, exactly as
>Invitrogen recommends.  
>

I have used supF as a tool to examine retroviral integration in vitro.  
These studies involved integrating a small linear DNA (supF flanked 
by 15 nt of viral U3 and U5 sequences) into pBCSK+, a plasmid from 
Stratagene.

Integrants were detected by transformation into MC1061/P3.  In
this system, selection with [Amp - 30, Tet - 7.5, Kan - 10] (all 
values in micrograms/ml final) resulted in selecting for integrants
in one orientation.

Dropping the selection to [Amp - 20, Tet - 5.0, Kan - 10] resulted
in detecting integrants in both orientations.

The results and our interpretation of them were published as
J. Virol. 70: 3571-3580 (June 1996).

Later,
Ashok
-- 
Ashok Aiyar
Department of Oncology            email: aiyar at ebv.oncology.wisc.edu
University of Wisconsin-Madison                  tel: (608) 262-6697




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