generating an empty vector!

[Sudheendra Rao N R]

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.

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