Phenol contamination - can't get the phenol out of my DNA sample (May/14/2007 )
I was wondering if anyone has ever had problems getting the phenol out of their sample after phenol/chloroform extraction. I extracted a digest twice with equilibrated phenol and three times with chloroform but I still smell phenol in the sample and when specking, I had a huge 270nm peak (which indicates phenol). I don't really know what could be going wrong. My phenol isn't old, or turned pink and I didn't think that chloroform went bad. Any suggestions would really help. The thing is this contamination prevents my sample from precipitating in alcohol and I can't run any PCR with it either.
You can try to do twice the cloroform extraction, and when you are pipetting try to dossolve your sample in 300 ul or more, then adjust the volume of your pipet from high volume to low volume. for example, first you take 100 ul until you reach a low volume adjust the volume of your pipet to 50 ul and lower volumes if necessary.
thanks, will try.
My phenol-chloroform extraction solution is a standard mixture of phenol, chloroform and isoamylalcohol. 25:24:1
Thus far I have not experience any problems with phenol removal. As standard practice, I ethanol percipitate my DNA after a phenol/chloroform extraction.
Ethanol percipitation is conducted with 3x volume of 100% ethanol with 1/10 volume 3M Sodium acetate.
Dextran can be added if the DNA quantity is small. THe sample is then spun down. The supernatant removed and the pellet washed with 70% Ethanol.
An optional step would be use pre-chilled 100%ethanol. (-80 Celsius). Or to chill the DNA-ethanol mix at -20 Celsius for 15mins or so.
Actually this may not come from the phenol, this may be a contamination by the silica or sugar compound present in a kit you use to purify your DNA.
I had a similar thing happening in DNA samples that never froze.
U can try to centrifuge the DNA when it's resuspended in water after chloroform treatment at high speed and p'recipitate the supernatant.
actually phenol contaminant only appear at 230 nm wavelength (check in maniatis) , so it's something else...
I thought 230nm absorbtion was salt? I'm pretty sure it is phenol that stays in my sample because it smells like phenol.....I'd know that smell anywhere.
you can try to dissolve your DNA in a 500 ul after phenol-cloroform extraction and precipitate with ethanol and sodium acetate, becouse if you have some residual phenol in solution, may be you can get rid the residual phenol in precipitation.
you may use too butanol for precipitating your DNA
just put 10volumes of it. vortex well
spin top speed 10'
if a lower phase remains, it's aqueous.. keep it and repeat the addition of BuOH, vortex, spin, till the lower phase disapear
wash well with etoh70% twice
let dry well
I prefer that as the butanol is more hydrophobic than etoh. Do it accepts more phenol and pellet better DNA than acetate ethanol
finally, it loads less salts than Na/ EtOH
As others suggested, I would precipitate the DNA and check the DNA conc. and use it for different experiments.