I am trying to amplify a plasmid (P3XFLAG-CMV-14 plus non toxic insert) through backterial transformation. The plasmid that I am using works great for transfection in HEK293T cells, however, after transformation and DNA extraction, the HEK cells don't express the protein at all (or at very very low levels).
I have sequenced my insert and the CMV promoter and they are fully intact.
I have linearized my construct and it migrates at the same rate as the transfectable one.
I don't believe endotoxin is a problem, since I've tried using DNA extraction kits with and without endotoxin removal buffer. Plus http://www.ncbi.nlm....pubmed/10997275
I have tried different chemical competent cells (DH5-alpha, JMH109 & XL10 Gold) without success.
I appreciate any inputs!!
Edited by Gangwolf, 09 January 2012 - 06:36 AM.