Phenol extraction - (Jul/15/2010 )
Sorry to answer so late,
Our phenol is yellow-ish becaus we add inside 8-hydroxiquinoline to prevent oxydation. It's fairly new, we bought it 1month ago and open it last week.
I will try phenol/chlorophorm only extraction also. Should-I do only twice ? Or try it more time to compense phenol extraction absence ?
Thanks for your help !
Sluvah on Jul 18 2010, 11:57 PM said:
Thanks for your help !
Sorry, I don't quite understand what you mean. Could you rephrase what you were saying?
Sorry for my english
My boss' protocol for phenol then Phenol/Chloroform then Chloroform extraction have 6 steps because each extraction (phenol, Phenol/Chloroform , Chloroform) are made twice.
For the Phenol/Chloroform and Chloroform extraction, should I do twice Phenol/Chloroform extraction then twice Chloroform extraction, or should I do it more times ? (Three time for example).
There should be a layer even with phenol only extraction, but it seems to be hardest to get.
? Strange protocol you have...
maybe its better to post the protocol here.
Here is the protocol :
(For archaea, but after the cell lysis, there shouldn't be any differences, no ?)
500ml archaea culture is pelleted and washed on N100 buffer (100mM Tris pH8, 100mM NaCl, 50mM EDTA pH8) then resuspended in 2 to 5 ml of N100 buffer.
Then, it's frozen at -80°C for several minutes then unfrozen at room temperature. The SDS is added (1% final) then the sarkosyl (0,8% final) (These two are probably too concentrated.)
After, 1 vol of phenol is added then mixed by gently inverting for 15 minutes.
Then, centrifugation 5 min at 4000RPM to separate layers. The aqueous layer is preleved, and a new phenol extraction is did (same thing, add a volume of phenol, mix, centrifugate).
Then, same thing but with Phenol/Chloroform (Two extraction)
Then, same thing but with Chloroform (Two extraction)
Then, precipitation with 100% ethanol, then with 70% ethanol.
After dessication, ressuspension of the DNA into the TE
Test of phenol absence by restriction enzyme digestion.
I think his protocol may work whis HIS strains, (streptomyces) but not with mine...
I plan to use an other cell suspension buffer (TE + NaCl 100mM) and try with and without the phenol-only extraction step..
Thanks for your kind help !
I think the problem you're having is due to using straight phenol, rather than buffer-saturated phenol. Prepare buffer-saturated phenol as shown under "Preparation of TE buffered Phenol" here.
I'm sorry, I forgot to precise. It's buffered saturated phenol, pH=8
I usually decide how many phenol-chloroform to do after looking at the first one: how is the interphase? Is the aqueous phase cloudy? Usually, 2 are plenty enough. I'm also surprised to see so many steps in your protocol.
My boss is cautious. REALLY cautious....
Fortunately, my boss is on vacation. So I will be able to freely adjust the protocol... Because the more I read about DNA extraction, the more I think just P/C + C extraction should be enough.
Thanks everyone !
Just to let you know,
I made a test with P, P\C then C extraction of a smaller culture volume with success. I think the error was too high concentration of detergent (SDS and sarcosyl) and a too low centrifugation speed (4000RPM, I haven't been able to find the rotor radius, so I can't convert this speed into "g". For a bigger volume, I will use another centrifuge where I can switch RPM to RCF)
My only problem is that I ressuspend my DNA pellet into too much TE buffer : It's too diluted, but I will re-precipitate it as soon as I can. I still need to check the absence of phenol residue, but I'm confident.
Thanks every one !