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Plasmid Prep Problem : gDNA - bacterial genomic DNA contamination in a plasmid minipreparation (Jul/01/2008 )

Hi There,

I've been a long-time and happy user of qiagen kits and I am now in a situation were I don't have access to such kits anymore... $$$... dry.gif

I am encountering problems with kits such as Fermentas GeneJet or the EZNA Kit (Biotek) and I always end up with a somewhat diffuse band above my ladder (e.g. >14kb), so I assume this is gDNA contamination. I used to make hand-made boiling preps before without any gDNA contamination and I am running out of ideas concerning where this gDNA contamination comes from, here are a few:

- do not overgrow liquid culture (16h in LB)
- start the liquid culture from a single colony (does it even matter?)
- do not vortex after cell lysis (I've followed the instructions very carefully, and I used to be less careful with better results)
- make sure the liquid culture is grown with enough aeration / enough shaking

Futhermore, after pelleting the bacteria, I resuspend them in buffer by pipetting, I used to do it as well without any problems with qiagen kits... does it matter now ?

thanks for the suggestions.


Overloading the columns can cause this. Try reducing the number of cells in your prep. You might also want to look at preparing your own silica binding matrix and using a "glassmilk" like prep, which is very cheap and gives column-like results.


Hi Veteran, thanks for your suggestions...

QUOTE (phage434 @ Jul 2 2008, 03:36 PM)
Overloading the columns can cause this. Try reducing the number of cells in your prep. ...

This wasn't the problem here, I tried again today with less cells, but still I had gDNA...
I'm kind of starting to think that it might be the shaker that is not shaking enough...
I will try qiagen kit to make sure now...

edit: the qiagen kit did not improve the results... I'll try to get rid of the gDNA with a PEG precipitation and post the results here...

edit 2:
as it turns out, I am still learning, and I guess a lot of people still are... I've been working with a few vectors out there, but it was the first time I was working with the pDRIVE vector (qiagen) and I guess because I used different kits and my liquid cultures were grown a bit differently than I had done before, I thought there might be a problem. In fact what I took for gDNA is probably the open circle form of my vector, running right above the top of my ladder, I never encountered that previously, but now I know I was doing anything wrong smile.gif


Confirmation by digestion (with 10 years old RE I managed to find in the lab...) It was the plasmid and not gDNA.