generating an empty vector!

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Mon Jul 14 23:15:34 EST 2008


Hello,
as far as remember, pcDNA3.1 does have a putative transcription start..wont
that make sure that tags are expressed even in empty vector which has got
self ligated..has any one tried detected overexpressed empty vector products
in a blot using V5 or His antibody?


On Tue, Jul 15, 2008 at 5:16 AM, Pow Joshi <pow.joshi from gmail.com> wrote:

> .
>
> 2008/7/14 Pow Joshi <pow.joshi from gmail.com>:
>
> >
> >
> > 2008/7/14 DK <dk from no.email.thankstospam.net>:
> >
> > In article <mailman.738.1216060696.3533.methods from net.bio.net>, "Pow
> Joshi"
> >> <pow.joshi from gmail.com> wrote:
> >> >2008/7/14 Kay Schink <k.schink_nospam_ from _nospam_gmail.com>:
> >> >
> >> >> 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
> >> >
> >> >
> >> >yes, I used to get large amounts of empty vectors as well....
> >> >self-ligated....and we did use them as controls.
> >>
> >> Poor control then. As I mention in another post, such a vector
> >> will not be expressing any protein with a 45 aa peptide that might
> >> very well affect results. E.g:
> >>
> >> http://www.ncbi.nlm.nih.gov/pubmed/11244567
> >>
> >> "The fusion of these epitopes with the recombinant proteins is not
> >> expected to alter the behavior of the protein of interest. In this
> report,
> >> we demonstrate that the mere expression of a cellular protein,
> >> hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr
> >> ligand, in two different vectors clearly altered its localization
> pattern
> >> in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA
> >> resulted in its nuclear localization, whereas the expression of this
> >> gene from a TOPO cloning expression vector, pcDNA3.1/V5/His,
> >> resulted in cytoplasmic expression. The native staining pattern
> >> of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34
> >> demonstrated cytoplasmic staining. During cloning, other leader
> >> sequences intended for targeting this protein into a cytoplasmic
> >> or a nuclear location were not fused to the actual ORF of this
> >> protein. Also, the amino acid sequence of the fusion region arising
> >> from cloning of hVIP/mov34 in both vectors does not match
> >> any reported NLS sequences. These results indicate that the
> >> choice of the expression vectors, as well as the position of
> >> synthetic epitopes, can significantly alter the behavior and
> >> the biology of recombinant proteins. This result suggests the
> >> need for a careful examination of these features when
> >> characterizing a newly identified protein."
> >
> >
> > WOW... thanks Dima .... Frankly, never bothered about it since we were
> > doing some specific IPs.... it seemed to work.... I see though that it is
> > definitely a concern.
> >
> > Pow
> >
> >>
> >>
> >> DK
> >> _______________________________________________
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> >> Methods from net.bio.net
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> >>
> >
> >
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