generating an empty vector!

Kay Schink via methods%40net.bio.net (by k.schink_nospam_ from _nospam_gmail.com)
Mon Jul 14 10:07:57 EST 2008


Sudheendra Rao N R schrieb:
> Hello,
> I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO vector.
> However i am not very sure about the control vector. In the TOPO TA kit, an
> expression control vector of pcDNA3.1V5/His6 LacZ was given however i cant
> use if for my experiments (it does some crazy stuff :( )..i did try pcDNA3.1
> without any V5 or His tag as empty vector and it seems to work fine..but
> just a doubt
> 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better
> control?
> 2. If yes then how to generate it? I guess i need not use T4 DNA ligase as
> TOPOisomerase itself can ligate after i use klenow
> 3. I beg for a Klenow protocol for this type of vector (could not find it on
> net)
> 4. Does V5/His6 tag make really big difference to the properties of a
> protein? if not then i can use empty pcDNA3.1 it self right?
> 
> kindly suggest..in urgent need
> 
> Sudheendra.
> 
How about using a vector obtained from a negative clone... If you do a 
TOPO cloning reaction, you usually get some clones that have an empty, 
self-ligated vector (at least with our Topo cloning kit you get it quite 
frequently ;-) ) These vectors should be empty and be a suitable control..

Good luck

Kay


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