21 replies to this topic

[Sluvah]

Hi everyone !

I'm new to phenol/Chlorophorm DNA extraction, and I tried today with my Boss and we get an interesting, but not wanted, result...
A milk-like solution, without aquaeous/phenolic difference after centrifugation...

What I did :
Ressuspend cells in 4ml of (100mM Tris pH=8, 50mM EDTA pH=8, 50mM NaCl)
Add SDS to final concentration 1%
Add Sarcosyl to final concentration 0,8%

Mix gently by inverting

Adding 4ml of pH8 saturated phenol
Mix several time by inverting


And I get this milk-like solution...
Phenol was yellow.


Any idea ?
Thanks for your help !

[pito]

You did do a centrifustep after the "mix several time by inverting step" , right?

[Sluvah]

Yes, 5min at 4kRPM (don't know the g value, need to calculate it)

[pito]

5 minutes might be too short.

and the speed has to be ok.

Maybe there is something wrong with the mixture at the start, you are sure the chloroform mixture is ok?  (phenol/chloroform/isoamyl alcohol 24/1 ratio?)

[Sluvah]

It was not the phenol/chlorophorm step yet, just saturated phenol.

We were planning to do phenole extraction twince, then phenol chlorophorm twice and to finish, chlorophorm only twice before washind with ethanol.

I note for next time to extand centrifugation time. How much do you suggest ?

[pito]

I am not really familiar with that protocol. But dont they use TE buffer when working with phenol extraction?

[Sluvah]

Don't know

I found in an article they are using TE buffer with 100mM NaCl added. I think I will try with this solution.

If anyone have a good idea, I will be pleased to hear it !

Have au good day

[Maddie]

Isn't yellow phenol, oxidized phenol?
Do you have a newer bottle?

[perneseblue]

Maddie, on Jul 15 2010, 08:51 AM, said:

Isn't yellow phenol, oxidized phenol?
Do you have a newer bottle?

yup, yellow and as it gets even more oxidized the phenol turns red.  

However if 0.1% w/v 8-hydroxyquinoline, a reducing agent used to protect phenol from oxidative damage was added, the phenol solution will turn bight yellow.

From Sluvah protocol I believe only a phenol solution is being used. No chloroform was added, so you can't obtained the aqueous-organic layer.

the composition of phenol/chloroform solution is

25:24:1 = phenol solution:chloroform:isoamyl alcohol

The phenol solution is composed of
10mM TE, 1mM EDTA solution saturated with phenol.

Add 0.1% w/v 8-hydroxyquinoline to your phenol solution, else it will get oxidised very quickly.


For DNA extraction the pH of the phenol solution is pH 8

For RNA extraction the pH of the phenol solution is pH 4.5

[pito]

perneseblue, on Jul 16 2010, 03:25 AM, said:

From Sluvah protocol I believe only a phenol solution is being used. No chloroform was added, so you can't obtained the aqueous-organic layer.

Thats where I was stuck, I couldnt understand it how he wanted to obtain that layer when using only phenol.

[Sluvah]

Hello

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 !

[perneseblue]

Sluvah, on Jul 18 2010, 11:57 PM, said:

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 !

Sorry, I don't quite understand what you mean. Could you rephrase what you were saying?

[Sluvah]

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.

[pito]

? Strange protocol you have...

maybe its better to post the protocol here.

[Sluvah]

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 !