transfection problem: DNA purity

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sun Nov 18 11:59:47 EST 2001


Dear Ricky, 
Clonetch rep. Macherey-Nagel to my knowledge offer endotoxin free plasmid 
kits. Sigma also should have some columns to remove endotoxins. Maybe also 
treating with periodate as you would do to cleave 1,2 diols present in all 
sugars could do the job. 
Maybe someone could comment on the latter, it just was something that came to 
mind.

You might ask PubMed or Google.

Regards, Wo

> Hi everyone,
> 
> I've a problem during transfection. I tried to transfect pcDNA-lacZ to
> mammalian cells using lipofectamine plus. When I used stock pcDNA-lacZ for
> transfection, everything worked fine and b-gal was obviously detected by
> western blot analysis. However, when I used pcDNA-lacZ which was prepared from
> Concert miniprep kit, no b-gal was detected. Then I tried to further purify the
> plasmid by phenol/chloroform extraction. The result was improved a little bit.
> Beta-gal was marginally detected. Such poor yield would be due to endotoxin,
> right? So, I wonder to know if there is a efficient way to remove endotoxin.
> Any comments and suggestions are welcome.
> 
> Thank you very much.
> Ricky  
> 
> 


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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.O.C.
e mail wolfsc at ibms dot sinica dot edu dot tw
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