Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Phenol extraction


  • Please log in to reply
21 replies to this topic

#1 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 15 July 2010 - 05:34 AM

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 !

#2 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 15 July 2010 - 05:37 AM

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

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 15 July 2010 - 05:39 AM

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

#4 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 15 July 2010 - 05:46 AM

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?)

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 15 July 2010 - 05:49 AM

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 ?

#6 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 15 July 2010 - 06:29 AM

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

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 15 July 2010 - 06:48 AM

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 ;)

#8 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 15 July 2010 - 08:51 AM

Isn't yellow phenol, oxidized phenol?
Do you have a newer bottle?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#9 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 585 posts
20
Excellent

Posted 15 July 2010 - 05:25 PM

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
May your PCR products be long, your protocols short and your boss on holiday

#10 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 16 July 2010 - 04:25 AM

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.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#11 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 18 July 2010 - 11:57 PM

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 !

#12 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 585 posts
20
Excellent

Posted 19 July 2010 - 07:40 PM

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?
May your PCR products be long, your protocols short and your boss on holiday

#13 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 20 July 2010 - 12:52 AM

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.

#14 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,491 posts
103
Excellent

Posted 20 July 2010 - 01:53 AM

? Strange protocol you have...

maybe its better to post the protocol here.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#15 Sluvah

Sluvah

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 20 July 2010 - 03:52 AM

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 -80C 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 !




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.