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Effect of a DNA prep on DE3

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#1 Dr_Watson



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Posted 10 November 2010 - 01:06 PM

Hi all,

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...

#2 perneseblue


    Unlimited ligation works!

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Posted 10 November 2010 - 08:28 PM

might there be a mutation in that particular plasmid
May your PCR products be long, your protocols short and your boss on holiday

#3 protolder



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Posted 10 November 2010 - 10:31 PM

Hola, I understand that you see your recombinant protein band before induction and adding IPTG the expression donīt increase. This is because there isnīt any repression of expression, levels of cAMP are high and the promoter/operator is open. Why do you try to repress the expression with glucose 0.1-0.2 g/l or better use a (DE3)laqIq strain? Good luck

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