Effect of a DNA prep on DE3
Posted 10 November 2010 - 01:06 PM
Here is my problem, I transform a plasmid into a cell containing the DE3 lysogen. The DE3 allows the expression of the T7 RNA polymerase after induction with IPTG. So I've transformed several plasmid into the cell and with one specific plasmid I don't see any expression of the T7 RNA polymerase after induction with IPTG.
I always have a control to check that the problem is not the IPTG or the Ab during the W.Blot. With different plasmids the expression is always of the same intensity. And that's what I don't understand as the type of plasmid shouldn't interfere with the DE3, should it?
So my question is: can the DNA preparation be the cause? I mean, if the preparation is not that pure (even though it's from a minikit) can it be a problem? (like using a too large volume of cells for the miniprep...)
thanks for any kind of help, suggestion, positive vibes...
Posted 10 November 2010 - 08:28 PM
Posted 10 November 2010 - 10:31 PM