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
- - - - -

Isopropanol precipitation problem


  • Please log in to reply
10 replies to this topic

#1 Macaronii

Macaronii

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 22 May 2009 - 01:30 PM

Hi all,
We started using a protocol for plant DNA extraction in which there is a step for precipitating the DNA from the samples using isopropanol. The problem is that after briefly mixing the isopropanol with the aqueous DNA suspension, a bubble forms at the bottom of the microtubes. It looks like if there are two phases, organic and inorganic.
Briefly, the protocol uses:
An extraction buffer (Tris, NaCl, EDTA), SDS, heating samples at 65 C, adding 5M potassium acetate, extraction with phenol:chloroform, chloroform, RNase, and then isopropanol. Spining time during the organic steps is for 3 min at max rpm. What could be forming the bubble, some phenol residue?
Any help is appreciated

#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 23 May 2009 - 08:25 AM

you should not be seeing any two phase after addition of IP. maybe you have touched the bottom phase during phenol:chloroform step. Becareful while removing the top phase into another tube.

do you see the DNA pellet after centrifugation?

#3 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 23 May 2009 - 10:22 AM

Go in with a pipette tip and draw the bubble off the bottom of the tube(s). Put it (them) in another tube and smell it -- is it phenol or chloroform?

#4 juanboja

juanboja

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 24 May 2009 - 09:58 PM

Are you adding the RNAse before the chloroform and then the IP to the top phase?

Edited by juanboja, 24 May 2009 - 09:58 PM.


#5 Macaronii

Macaronii

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 25 May 2009 - 09:25 AM

Curtis: No pellets were seen after adding IP to the upper phase and centrifugation.
I did as HomeBrew said, and the bottom bubble smells like phenol!!
Juanboja: I transfered the upper phase to a clean tube after the chloroform step, and then added RNase.

It appears that I am taking phenol after the phenol:chloroform step!! Well, I have done this hundreds of times using other protocols without problems....It seems that I'm getting old ;)
Thanks a lot guys

#6 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 25 May 2009 - 11:19 AM

If your final step is an isopropanol precipitation, does the trace of phenol matter?

#7 Macaronii

Macaronii

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 25 May 2009 - 11:35 AM

After adding the IP, mixing, and centrifuging at 13k rpm for 5 min, a DNA pellet should be present. Instead I have this bubble at the bottom. Could it be possible that a longer centrifugation is needed after the phenol:chloroform step? The protocol calls for a spin at 13K rpm for 3 min.

#8 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 26 May 2009 - 04:41 AM

With phenol it is sometimes possible to have an layer inversion if the aqueous phase is dense due to very high salt concentrations. You think you are taking the aqueous layer, but it is on the bottom rather than on the top. This never happens with phenol/chloroform, where the chloroform density is quite high. Might this have happened to your samples?

#9 Macaronii

Macaronii

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 26 May 2009 - 07:21 AM

High salt concentration in the sample...it could be possible. Before the phenol:chloroform step, 94 ul 5M potassium acetate are added to the mixture of ground tissue, extraction buffer and SDS, kept on ice for 5 min and then spin at 13k rpm for 5 min. The supernatant is then transfered to a clean tube and the phenol:chloroform mix is added. What is the purpose of adding potassium acetate? In some protocols I have used 7.5 M ammonium acetate.

#10 lsek

lsek

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
2
Neutral

Posted 29 May 2009 - 07:24 AM

I thought "bubbling" happens frequently after addition of isopropanol (IP) to the lysate? I observed the same thing with EtOH.

In genomic DNA precipitation, addition of alcohol will frequently result in "aggregation" of DNA to form thread that you can see with naked eye after inverting the tube to mix. I see this most of the time.

I'm assuming that the potassium acetate (or high salt) used here is for salting out proteins and the organic solvent is to enhance the sequestration of protein from the aqueous phase.

Just curious, you're extracting DNA from plant material? Is it from fruits or leaves? I used to extract DNAs from both fruits and leaves and it's a bummer to avoid polyphenol (or phenolic compound) contamination as well as its oxidation that will irreversibly bind to DNA and causing unretrievable or unusable DNA for downstream application like PCR etc. Heating, it seemed, exacerbates this oxidation. I remember that I dreaded using reducing agents such as beta-mercaptoethanol to avoid such oxidation. However, some papers used PEG, CTAB method etc....

Hope this input will be of some help.

Edited by lsek, 29 May 2009 - 07:25 AM.

===><===


#11 Macaronii

Macaronii

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 10 June 2009 - 11:08 AM

Just a little update on our isopropanol problem. It seems that Isek was right on the point regarding contamination with polyphenol compounds in the sample. We are trying to extract DNA from field grown tomato plants showing strong viral symptoms (stunting, leaf curling, mosaics, leaf deformations) for viral identification using PCR. The "bubble" appears during DNA extraction of these samples. For comparison, we used cotyledon leaves from cucumber seedlings and the bubble was absent!. We are going to try some modification to the protocol or use a different one:)
Thanks for the input...

Tubes on the left show the "bubble" (tomato), samples on the right without "bubble" (cucumber seedlings)
Posted Image




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

©1999-2013 Protocol Online, All rights reserved.