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Culture Grow well but No Plasmid


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#1 DNA

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Posted 05 January 2010 - 05:38 PM

Hi,

I recently transformed my plasmid in PCDM8 vector using both ampicillin and Tetracycline.
It did work, culture grow well but when I do miniprep, no plasmid are found on gel though the nanodrop gives me conc of 128ng/uL.
I have successfuly transformed this in the past , now I dont know what is wrong?
Can someone explain plus what the nanodrop is reading then, the genomic DNA?

Hope to have a quick response.

#2 pDNA

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Posted 06 January 2010 - 10:45 AM

maybe sheared genomic DNA? ...but you should see such a concentration somewhere in the gel! (maybe as a black spot in the pocket where you loaded your sample or as a smear along the lane?)

You did some negative control, just electroporating water? Maybe your competent cells are faulty, or your antibiotics are degraded due to too long storage.

Try to pick some clones from the orginal plate and streak them on a fresh plate ...do they grow again?

Regards,
p

#3 DNA

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Posted 06 January 2010 - 04:30 PM

maybe sheared genomic DNA? ...but you should see such a concentration somewhere in the gel! (maybe as a black spot in the pocket where you loaded your sample or as a smear along the lane?)

You did some negative control, just electroporating water? Maybe your competent cells are faulty, or your antibiotics are degraded due to too long storage.

Try to pick some clones from the orginal plate and streak them on a fresh plate ...do they grow again?

Regards,
p


Yeah I can that concentration as diffused band on teh top of teh 1kb ladder.
Antibiotics old, I dont think so as I have simultaneosuly done a couple of other receptors protiens and I got nice miniprep and RNA of that.

So I think I should do the negative control amd see what happens...




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