Standard Chloroform Phenol Extraction Questions? - (Jul/22/2005 )
Hi,
I am currently cleaning up a large batch of DNA samples using a standard chloroform:phenol:isoamyl alcohol cleanup 25:24:1. Using a spectrophotometer, specifically a genequant, to measure my 260/280 optical densities pre and post cleanup I am really not seeing a positive change. My 230/260 ratio is still <1 but in some cases my 260/280 ratio is actually dropping from 1.5 to 1.2, 1.1 and lower. I don't understand whats going on, this has happened to the last 5 DNA samples (thankfully I'm cleaning up spare samples atm to test the method) which were cleaned up @ 2 different times with the same method.
The protocol that i'm following is as follows:
Starting with a DNA volume of ~20 microliters TE.
1) Rnase treatment (stock 10mg/ml) 1 uL for 30 minutes @ 37 C
2) Proteinase K treatment (stock 20mg/ml) 1 uL for 1 hour @ 50C
3) Bring up sample volume to 100 microliters in TE
4) Add equal volume (100 microliters) of 25:24:1 chloroform:phenol:isoamyl alcohol. Mix vigorously.
5) Spin @ max speed for 10 minutes @ room temp to achieve layer separation.
6) Transfer aqueous layer to new tube and add 1/12 volume of 5 M NaCL, mix vigorously and let stand at room temp for 5 minutes.
7) Add 2.5 volumes of ice cold ethanol to sample and leave @ -80C for 30 minutes.
8) Spin @ max speed for 10 minutes @ room temp
9) Decant supernatant and wash DNA pellet with 75% EtOH.
10) Resuspend pellet in a volume of TE.
11) Place sample @ 50C for 10 minutes to help complete solubilization
Is my 260/280 ratio just being unreliable? or is something wrong with this protocol?
-CW
Are you drying the ethanol off of the samples before resuspension? I typically do 2-3 minutes in a dessicator to remove residual ethanol. Ethanol can also be removed by leaving tube open on the bench for a longer period of time (evaporation) but I am not sure how long is necessary for complete removal...
We also use sodium acetate as the salt, the acetate is supposed to be removed more easily...
I also typically use 2 volumes of ethanol to precipitate DNA and 2.5 volumes for RNA precipitation. Not sure why or if this would make a difference.
I think you should be doing a chloroform extract following the phenol/chloroform extraction. Otherwise, you often see phenol contamination of the samples, which inhibits many enzyme reactions. Also, the phenol affects the UV quantitation (looks like more sample than is really present).
I also use 1/10 th volume of 3M sodium acetate rather than NaCl, but I'm sure the salt works also.
It probably is somewhat harder to remove with the 75% EtOH wash.
What do yo want to do with the DNA? It might already be clean enough for your purposes.
Also as mentioned above use NaAc rather than NaCL and just do the ETOH precipitation at room temp. Low temperatures won't help the precipitation and will alone cause more of the salt to be brought down.
i tratd my DNA samples in 500µl (for RNase and prot k). I am wondering if increasing the volume probably enhance furhther steps of purification.
after adding absolute ethanol, may i suggest you to spin 30' rather than 10' ? i've noticed differences and better ratio...
for danoel :
I agree if you use butanol instead of ethanol. But cool the sample increase the precipitation in case of ethoh precip...
Centrifugation and precipitation time has more of an impact on DNA yield than temperature IMO.
If you have very low concentration of DNA and you need to get near 100% recovery (and don't want to use a carrier like glycogen), then I have found leaving the EtOH precipitation o/n at RT in the dark before centrifuging for 20-30 min works very well.