4 replies to this topic

[ram]

Hello..
I am using pCR T7/NT-TOPO expression vector for expressing a plant gene. As a negative control I have to have empty vector expression. I have read somewhere that linear empty TOPO vectors cannot be transformed since they have topoisomerase at the cloning site. How can I then prepare E.coli cells harboring empty TOPO vector?
If I get such cells prepared by some means, can the isolated TOPO plasmid be digested in some way and used as a expression vector for ligation using ligase enzyme?
  

Edited by ram, 02 January 2010 - 02:13 AM.

[phage434]

If you are unlucky (i.e. most of the time) you can count on having several colonies of your TOPO cloning transformations contain short or non-existent inserts.  You can save one of these for a control.  But the transformation control really can be almost any vector, though it might be a good idea to choose one with the same antibiotic resistance and origin incompatibility group.

It is difficult to prepare TOPO vectors straightforwardly in the lab, one of the reasons Invitrogen makes so much money off of them.  This would not be the first project I would undertake, though I would be interested in hearing from anyone who has succeeded in doing this.

[HomeBrew]

Or, perhaps an even better control (if you're looking for a control for your expression experiments) might be to save one of your clones that contains your insert, but oriented the wrong way for the vector-borne promoter you're using.  Then your positive and negative controls have the exact same size, DNA, etc. This assumes, of course, that there's not a promoter on the other side of T7 that will be active in your recipient strain (I don't recall the map of pCR T7/NT-TOPO expression vector).

[ram]

Thanks for the comments!
That's a great idea HomeBrew! In fact, I have a clone in which the insert has been reverse oriented. But as far as I understand, the reverse insert should not have complete open reading frame. Otherwise the expressed protein would have the size approximately same as the test sample, isn't it?

[HomeBrew]

That's true, if I understand your point -- if the result of your correct clone produces a fusion protein, the oppositely oriented insert won't have the part of the fused orf contributed by the vector.