Plasmid DNA problem - PEG precipitation (Oct/12/2006 )

I am trying to isolate plasmid DNA for transfection studies. I have been following the protocol from Sambrook et al (Molecular Cloning) for midi size cultures (200ml). I can isolate plasmid DNA fine, but following purification with PEG, I lose most if not all of my DNA. I saved the supernatant from the PEG step, and can precipitate the DNA back with EtOH. Is there a way to purify DNA aside from PEg and CsCl gradient? Also, is it necessary to purify the plasmid for transfection?

Any suggestion or ideas would be greatly appreciated.
Thanks,
Dustin

-dval7780-

The PEG problem is caused by getting the PEG volume : DNA concentration wrong. If the DNA concentration is too low, it won't percipiatate in PEG. Similarly if the DNA concentration is too high, the PEG+DNA solution becomes too dense and only a small percentage of the DNA percipitates.

It is a little bit of a trick to guess the "right" amount of PEG to add.

As for transfection... what kind of transfection are you doing? It would help clear things a bit. Although generally, PEG percipitation is conducted to clean up the DNA... I have never tried running without PEG percipitation. It might work (I don't know)... and all the extra DNA bit is just acts as carrier....

-perneseblue-

followed discussion :
mail from dval7780

QUOTE

I start with 200ml of culture, spin down, wash; spin; and resuspend in 50mM glucose, 25mM Tris, 10mM EDTA. I then add 2ml of lysozyme followed by alkaline lysis. I then add 5M KOAc, 3M acetic acid, then spin. collect supernatant and precipitate DNA with isopropanol. wash with 70% EtOH and resuspend in 3ml of TE. I then add 5M LiCl, spin and save supernantant. I add isopropanol; spin; and wash with 70%EtOH; add 500ul TE with Rnase A for 30 min, then add 500ul of 13% PEG 1.6M NaCl. Spin then do phenol-chloroform extraction and EtOH precipitation. By then I have NO pellet whatsoever.
Any suggestions on changes in my extraction?
Thanks again.

-fred_33-

as your culture is 200ml allvolumes should be adjusted to it.
So you've to adjust for RNase treatment : at least 2ml of 10mM Tris pH 8 100µg/ml RNAse A
then do a proteinase K (important for qualit prep and reduces endotoxin).
phenol chloroform the preparation.

Save the aqueous phase. Add 10µl Na Acetate 3M pH 5.4 vortex briefly and add 1ml EtOH 100%
pellet should start to form.
Then spin 10' full speed
Resuspend in Tris pH 8
For 500µl tris used add 200µl 30%PEG NaCl 1M, solution which should be good homogeneizd by magnetic swirl for 5' before using (i homogenize at same time i resuspend my plasmid)
Invert 4-5 times and put in fridge overnight.
Then spin 30' full speed (pellet may not be visible, and i save also the supernatant)
wash with 70%etOH
resuspend and quantify

So changes are the PEG concentration and the fact of an overnight pelleting.

-fred_33-