generating an empty vector!

[Pow Joshi]

.

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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