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-Chloroform Extraction (DNA)

DNA extraction Phenol-chloroform genotyping

  • Please log in to reply
5 replies to this topic

#1 Mad researcher

Mad researcher

    Researcher in making

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
7
Neutral

Posted 31 October 2013 - 12:28 PM

Hi again,

 

I am looking for a protocol for phenol-chloroform extraction of DNA from tissue (rat tail). I found a lot of protocols online but none of them specifies the extraction procedure. They more likely give the purification steps. 

I want to know how do i treat the tissue for DNA extraction.

 

Looking forward

 

Cheers,


Edited by Mad researcher, 31 October 2013 - 12:31 PM.

Cheers,

Mad Researcher

#2 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,531 posts
99
Excellent

Posted 31 October 2013 - 12:36 PM

here are several: http://www.protocol-...tion/index.html


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#3 Mad researcher

Mad researcher

    Researcher in making

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
7
Neutral

Posted 31 October 2013 - 12:40 PM

Thanks mate.

 

I had a very quick question. Can i substitute ethanol for Isopropanol (we ran out of isopropanol).

 

Cheers


Cheers,

Mad Researcher

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,471 posts
247
Excellent

Posted 31 October 2013 - 01:26 PM

If this is the DNA precipitation stage, then yes, it can be substituted. For isopropanol, you need only 0.6 volumes for precipitation. For ethanol, you need to add 2.5 volumes, so you need a larger tube to spin the precipitate in (or you can divide it).



#5 Mad researcher

Mad researcher

    Researcher in making

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
7
Neutral

Posted 31 October 2013 - 01:31 PM

  I am talking about the 7th step in this protocol

 

Cheers,

 

Obtain the last 1 to 3 mm of the mouse tail and place directly into an eppendorf. 

2. Add 200 μL of Digestion buffer. 

3. Add 2 μL of Proteinase K (10 mg/mL) [] 

4. Stir at 800 rpm for +/- 6 hours or 600 rpm overnight, both at 55 ºC. 

5. Centrifuge at 14000 rpm for 15 min. 

6. Remove supernatant (≈ 180 μL) and put it in a new eppendorf. Note: while you remove it, avoid bring together mouse piles. 

7. Add 180 μL of isopropanol (volume 0,7). Note: isopropanol is easily evaporated, so do not let flask open for big time. 


Cheers,

Mad Researcher

#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,471 posts
247
Excellent

Posted 31 October 2013 - 03:05 PM

Yes, instead of adding 0.7 volumes of isopropanol, you  can add 2.5 volumes of ethanol.







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

©1999-2013 Protocol Online, All rights reserved.