HELP No DNA after Nucleic Acid Extraction (Qiagen kit) - (Aug/21/2008 )

Hi to everybody.
I have a problem and badly look for solution.
I am trying to extract plasmidic DNA from E. coli DH5alfa strain (from bacterias both freshly transformed and glycerol stocks). My O/N cultures looks ok, the bacterias grow well. Then I use Qiagen kit (Midi). I have a nice white pellet after lysis. However, I obtain no DNA pellet after isopropanol and thus no DNA at the end.
I have changed the kit (different lot or even different supplier ie Roche, changed Midi for Mini kit), changed isopropanol, antybiotic stock (it happens when I use kanamycin and ampiciline), LB, still nothing. The only thing that occurs to me is that I have an environmental contamination that grows with my bacterias but in this case it would be resistant to both kanamycin AND ampiciline. However, when I leave opened LBagar plate with antibiotic or liquid LB with antibiotic and then incubate it O/N at 37ºC nothing grows.
What is happening? Im going crazy...
Thanks a lot.

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Have you done Isoprop-precipitation before, are you accustomed with it? If you do not mix the DNA with the Isoprop, you will not precipitate any DNA and get no pellet, could this be the problem? Otherwise.... I don't know.... sounds strange

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I'm a bit confused here. You say after lysis you have a nice white pellet, what do you do then?

This "nice pellet" is actually all the bacterial proteins and other crap you dont want, your DNA will be in the lysate (ie the supernatant generated during the lysis step), that's what you need to add to the column, elute and isopropanol precipitate.

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I am accustomed with it. I was always working with Qiagen kits (for about 5 years). Problem is that NOBODY in the lab can obtain any DNA so it´s not that I am the only one who does something wrong.

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The presence of a white pellet after the lysis doesn't usually tell you if lysis was good or not. Typically what I would look for is that the solution turns clear after adding the lysis buffer, P2, but before buffer P3. Bacteria other than E.coli will typically have a harder time lysing and the solution will not turn clear. Also bacteria other than E.coli have a different smell than normal E. coli and this is what I usually use to help determine if contamination is a problem. You could also determine if your plasmid is present in your bacteria by doing a PCR reaction on some bacteria itself.

Are you doing the midi prep because you have a low copy plasmid, or because you want to bulk up a high copy plasmid? If bulking is the reason, then I would definitely do a miniprep first to see if that comes out ok, then try the midi prep. If its because its a low copy plasmid, you could also try doing a miniprep, although depending on just how low copy it is you may or may not see any DNA (I would heat your elution buffer to 55C if you decide to do a miniprep with a low copy plasmid as this will better help you to obtain DNA). For low copy plasmids or really saturated bacterial cultures you may need to increase the lysis buffers (midiprep) accordingly to ensure that you get good lysis of your cells. If you are certain that contamination is not an issue, then I would go ahead and do a gel analysis of the entire procedure to see where you are losing your DNA. This is described in the protocol book - basically what you do is save some of each fraction and precipitate it with isopropanol, then run it on a gel.

Hope this helps,
smu

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Of course, I add the lysate to the column and precipitate it with isopropanol.
By "nice pellet" I wanted to say that apparently I have no problems with the lysis of my bacterial culture which could be responsible for my problem. Unfortunetely it´s not the case...

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Sorry, didnt want to sound so horribly patronising Having a bit of a bad day....

If no one in the lab is getting DNA might be worth checking your buffers, specially the pH. Also try with really cold isopropanol in addition to the warm elution buffer suggested by smu.

I agree with smu in the mini-midi comparison. Finally, you could also split your culture and do several mini-preps and the pool your DNA.

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Thanks a lot.
My solution does turn clear after adding the lysis buffer. I have a high copy plasmid (pET23b). I obtain the same result (no DNA) with mini and midi. Until now I was quite sure that the plasmid is present because I was using a glycerol stock to run my cultures (previously I could obtain decent quantity of DNA using the same tube of glycerol stock). We always prepare two tubes of glycerol stock. One to work with and the other one "untouchable", both kept in different freezers. I tried to extract DNA from the "untouchable" one in case if the working one got thawed and bacterias died. Still nothing.
However I will do PCR just to make sure that the plasmid is present. Thanks a lot.

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Thanks a lot. I will try with cold isopropanol and taking aliquouts at each step to run a gel.

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Hmm...starting to sound more strange being that it was working before and now its not. Have you tried streaking out your glycerol stock onto plates? Are you sure that there isn't DNase contamination somewhere in your lab - are preps from other plasmids turning out ok? Are other people having problems?

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