plasmid extraction

[molecular_micro]

Hi,

I've been trying to extract plasmid DNA from 2 E.coli host strains and 1 lactococcus lactis host strain. I am using a QIAprep mini kit by qiagen and am following the protocol as directed. Upon taking a nanodrop reading I found I had extremely low yields and poor purity. I'm new to molecular microbiology and this is a completely new technique for me. I've read many troubleshoots and have addressed most of the common suggestions (not lysing cells for more than 5 mintues, ensuring the culture has been grown in the presence of the correct antibiotic, thorough (but gentle) mixing, etc.). Furthermore, when I run gels with 10ul of my final elute I am seeing no bands (or faint to the point of being barely visible).

Can anyone offer any further suggestions which may improve both yield and purity from this kit.

Also, excuse my potentially stupid question, but I'm assuming that with plasmid DNA I am looking for the same 260/280 and 260/230 ratios I would be looking for with a gDNA extraction to indicate purity?

Thanks in advance for your suggestions.

[relaxin]

First of all, what are the plasmids you want to purify? Some are low copy number plasmids, so the yield will be low. You can increase the volume of overnight culture. The protocol is designed for 3 ml overnight culture. If you increase to 6 ml, then you need to double the volume of P1, P2, and P3 buffer. Alternatively, you can do it in two tubes each as described in the protocol. First load the lysate from one tube, wash with QC and elute DNA with QF. Then re-equilibrate the spin column with QBT, and process the second tube of lysate. Combine the eluted DNA and concentrate by ethanol precipitation.

[mchlbrmn]

On the positive side, your OD and electrophoresis results are consistent.
I don't know; everything sounds good. You're right that a common problem would be growing the cells without antibiotic, if you haven't you can double check the concentration (100ug/ml ampicillin is common, and may be 1/10 this for some others) and that you have the correct antibiotic. Yes, the OD ratios should be the same for genomic and plasmid DNA, and when the total amount is very low the ratios become inaccurate. You should have had a size marker ladder to show the gel and staining was working well. You might call a Qiagen tech help line. Part of the price you pay is for this assistance, and speaking directly they may find a problem. The only thing that comes to mind is that there are no plasmids in the cells you are growing, they could have lost the plasmid if previously grown without antibiotic, or it could be a contaminating bacteria (err, not likely for 3 strains).
Oh, if the host bacteria are not recombinant minus mutants, maybe the antibiotic selection integrated into the genome and the plasmid was lost. Common cloning E coli strains should all have rec minus mutations.

[memari]

The problem comes from P2 buffer. It has 200 mM NaOH and 1% SDS .
NaOH is decayed by CO2 of air during opening the bottle.
Prepare it freshly yourself.

http://openwetware.org/wiki/Qiagen_Buffers

http://bitesizebio.com/articles/the-basics-how-alkaline-lysis-works/

http://www.protocol-online.org/forums/index.php?app=forums&module=forums&section=printtopic&client=printer&f=1&t=25610

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Babak Memari