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.
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....
followed discussion :
mail from dval7780
Any suggestions on changes in my extraction?
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.