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
- - - - -

How to get rid of DNA contamination in RNA prep

DNA contamination rna

  • Please log in to reply
4 replies to this topic

#1 mike2k

mike2k

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 22 August 2013 - 06:50 AM

Hi everybody,

 

I´ve a problem with contaminating gDNA in my RNA extractions. I follow a Hot SDS-Hot Phenol protocol and I end up with approx. 30 -50 % DNA regarding the total amount of nucleic acids (This is an estimation by interpreting a reducing agarose gel). I´ve checked and double checked the pH of the Phenol, which is around 5. Furthermore I add NaOAc to decrease pH during the extraction - so I don´t think it´s the pH. But what can it be then? I´ve done up to 4 Phenol extractions with a single sample getting the same result. Furthermore I´ve tried to do the extraction with Phenol:CHCl3:IAA (25:24:1). However, there it seems that there is even more DNA in it in the end.

 

TRIZOL extraction works better, though. However the overall RNA yield is much lower and TRIZOL is expensive. So I´d rather like to stick to the Phe:CHCl3 protocol which gives good quality RNA when analyzed with agilent Bioanalyzer.

 

I´ve read now dozens of RNA extraction protocols, but I cannot really find a solution for my problem.

 

Can anybody help?

 

thanks for every response


Edited by mike2k, 22 August 2013 - 07:03 AM.


#2 PhalanxBio

PhalanxBio

    member

  • Active Members
  • Pip
  • 18 posts
3
Neutral

Posted 22 August 2013 - 08:34 AM

Probably not the answer you're looking for but I suggest going with Trizol.  You say the results are better but the RNA yield is lower.  Perhaps the RNA yield only appears lower because there is no contaminating DNA.



#3 mike2k

mike2k

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 22 August 2013 - 10:25 PM

thank you for your reply! In a direct comparison on a gel the RNA yield with TRIZOL is considerably lower. However, my problem is that you really need large quantities of TRIZOL, which is quite expensive and still there is visible DNA contamination on the gel. If if have too I will use TRIZOL, however I won´t if there is any other way.

 

Is it possible to "overload" the phenol:CHCl3 System? After lysis with SDS the solution becomes extremely viscous. I have doubled the amounts of all reagents in the original protocol, however without success so far. Even if not, after 3-4 extractions the DNA should be washed out, shouldn´t it?

 

 

thanks



#4 memari

memari

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 148 posts
11
Good

Posted 23 August 2013 - 02:03 PM

Use RNAzol which costs half. I prefer it. Both(RNAzol and  TRIZol) are  of inventions of Chomczynski.

 

http://www.mrcgene.com/rnazol.htm

200 ml = 203.00 $

 

compare with  TRIZol.

http://www.invitroge...l?CID=fl-trizol

200 ml  = 416.00 $


Edited by memari, 23 August 2013 - 02:06 PM.

-----
Babak Memari

#5 memari

memari

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 148 posts
11
Good

Posted 23 August 2013 - 02:17 PM

Make Trizol yourself:

 

1- Saturated Phenol with Tris.HCl.pH 4.8 to 5

2- DTT  1 %

3- Guanidine Isothiocyanate 4 Mol 

 

You can also try adding Triton-x-100 or NP-0 from 0.1 to 0.5 %.


-----
Babak Memari





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

©1999-2013 Protocol Online, All rights reserved.