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plasmid concentration and freezing - (Dec/14/2007 )

I extracted plasmid from DH5A cells using Promega PureYield Midiprep from 100 ml broth following the exact protocol. Elution was done in 500ul water. The extracted plasmid does not seem to freeze even after a week at -20. So I thought that they might contain some remaining ethanol from buffer although I added another centrifugation step to minimise this from happening. I checked the plasmids on gel but a clear band was observed. Also I measured concentration using spectrophotometry also to see for any impurities in it and as expected the concentration was very high 500ug/ml (Promega says 250ug from 50ml broth) I also had 260/280 more than 1.8 and also 260/230 of 2.2.

does the high DNA concentration affects freezing?

any recommendations whether I need to clean the DNA using microcon filters. I need this DNA for transfections into HeLa and COS-7

Thanks.

-dnalab-

Your DNA conc is 500ng/ul in a total of 500ul. Its not a very high conc..

You could precipitate the DNA to get rid of the ethanol and resuspend the DNA in any volume you need. use TE to resuspend maxi prep DNA. Its better than water.

-scolix-

QUOTE (scolix @ Dec 14 2007, 02:38 PM)
Your DNA conc is 500ng/ul in a total of 500ul. Its not a very high conc..

You could precipitate the DNA to get rid of the ethanol and resuspend the DNA in any volume you need. use TE to resuspend maxi prep DNA. Its better than water.



Yes I do not think that 500ng/ul is a high concentration and so I suspect in ethanol. Thanks for info.

-dnalab-

If you think it's ethanol: do you have a speed vac? Apart from this, your absorbance doesn't seem to indicate contamination from ethanol.

-vairus-

QUOTE (vairus @ Dec 15 2007, 12:59 PM)
If you think it's ethanol: do you have a speed vac? Apart from this, your absorbance doesn't seem to indicate contamination from ethanol.

It's definitely the ethanol. I've tried those Promega kits and had the exact same problem: ethanol in the eluate, even after drying the bejeesus out of the column. Everything looked fine with the spec and ont eh gel, but it didn't freeze at -20°C. I suspected something was up when I eluted with 500µl water and 550µl of liquid came out of the column. To be honest, I've tried the Promega Midi kits again a few times, and have given up on them and gone back to Qiagen. I even wrote a letter of complaint to my local Promega rep due to my extreme dissatisfaction.

To salvage your current midiprep DNA, ethanol precipitate it.

Ginger

-Ginger Spice-