5 replies to this topic

[Gangwolf]

Hi all!
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.

[jws]

At first, you need to confirm that the transfection efficiency is good. To do this, plasmids containing indicators like GFP can be mixed with your vector in a ratio like 1: 10 and transfected together. After checking cells with fluorescence, you can know how efficient the transfection is.

Second,  you need to check if the corresponding mRNA is well expressed in cells using real-time PCR.

The third is that you need to check if the protein is stable inside cells.

HEK293T is insensitive to the toxicity of various transfection reagents. It is also recommended that you can increase the stringency of transfection like more lipofectamine.

[Gangwolf]

I have done that  
Transfection efficiency is around 70% with GFP.
The expression of my construct at mRNA level is high and the construct that cannot be expressed is at the same level as my negative control empty vector.
In terms of stability, my protein can be detected by WB a week after transient transfection.

I have compared Lipofectamine, Turbofect, Exgen and JetPEI transfection reagents, where Turbofect is clearly the best.
Transfection itself is not the problem in this case. The question is why does transformation of a fully transfectable plasmid renders it non-transfectable?

Thanks for your comments!!

[jws]

Have you tried transfection of linearized plasmids?

[Gangwolf]

That I haven't tried! The plasmid that works great before transformation is not linearized, so do you really think it would make that big of a difference for transient transfection?

[jws]

I don't know, too. Just speculation about intercalacted DNA.   Is it possible that your condition is due to DNA modification in bacteria?