protein leftover in DNA - how to get rid of it? (Jun/30/2006 )
After using the "EndoFree Plasmid Maxi Kit" from Qiagen, I ended up with a white pellet. Salts I thought and did a ethanol precipitation. The pellet looked better after that, but I lost a third of my DNA. Now I measured the A260/A280 ratio in a photometer and this showed me, I had lots of proteins in my DNA (Ratio=1.3). I want to use the DNA for transfection, so I need it really clean. Any suggestions what I should do? A woman from Qiagen told me not to do a RNAse treatment, as it would cost me lots (if not all) of my DNA. I have 30 µl of DNA (aqueous phase) (concentration: 8.4 µg/µl) can I do a phenol-chloroform thing with this small volume/samll amount of DNA?
You can do a phenol. The concentration is quite high, therefore you could even dilute it beforre the phenol but I don't think there is any controindication to a phenol extraction. What do you think, experts from the forum?
add 1Volume of phenol chloroform alcool isoamyl (25-24-1)
mix gently by inverting the tube (do not vortex).
centifuge 10 min at 12000rpm
carefely remove the supernatant (aquouse phase) when therse is your DNA without protein and you can verify by OD 280nm
Actually, I'd recommend that you at least wash with chloroform to remove residual phenol, which is highly inhibitory to downstream applications and also affects spectrophotometer readings. I would dilute this DNA 10-50x before I started to 100- 500 ng/ul, phenol chloroform extract once or twice, extract with chloroform, and then ethanol precipitate. Vortexing plasmid preps is usually not a problem.
'phage434' is right but I'M not OK for vortexing DNA