generating an empty vector!
(by pow.joshi from gmail.com)
Mon Jul 14 12:09:37 EST 2008
2008/7/14 Kay Schink <k.schink_nospam_ from _nospam_gmail.com>:
> Sudheendra Rao N R schrieb:
>> I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO
>> However i am not very sure about the control vector. In the TOPO TA kit,
>> 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
>> 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
>> 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
>> 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
>> 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.
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