Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > DNA Techniques > DNA Extraction Forum
Register Search Today's Posts Mark Forums Read

DNA Extraction Forum DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Combining Trizol DNA extraction with QiaAMP micro purification

Combining Trizol DNA extraction with QiaAMP micro purification - DNA Extraction Forum

Combining Trizol DNA extraction with QiaAMP micro purification - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-30-2009, 08:36 AM
Pipette Filler
Points: 88, Level: 1 Points: 88, Level: 1 Points: 88, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Combining Trizol DNA extraction with QiaAMP micro purification



Hi!

I'm working on a project were I extract RNA from frozen breast tumortissue, using TriZOL for microarray analysis. I'm interestet in a protocol were I can extract DNA suitable for arrayCGH from the remaining lower/interphase.
I've done some testing were I've extracted DNA using the Back Extraction Buffer described by Ambions TRI-reagent and then done a clean-up using Qiagen qiaamp micro column. I manage to get 260/280 ratios around 1.8.

I also want to add a RNase step, but how and when do I do that?
Does anybody have experience with something similar og have any ideas to improve the protocol?

Best regards
Zeus
Reply With Quote
  #2  
Old 06-30-2009, 04:32 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: Combining Trizol DNA extraction with QiaAMP micro purification

You should be fine using the Back Extraction Method. Such as here --> [Only registered users see links. ]

Once your DNA is in TE or water you can perform the RNAse digestion, then take OD measurement.
Reply With Quote
  #3  
Old 08-07-2009, 10:01 AM
Pipette Filler
Points: 88, Level: 1 Points: 88, Level: 1 Points: 88, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Combining Trizol DNA extraction with QiaAMP micro purification

Quote:
Originally Posted by danfive View Post
You should be fine using the Back Extraction Method. Such as here -->
Once your DNA is in TE or water you can perform the RNAse digestion, then take OD measurement.
I have actually tried exactly that protocol you are linking to. But I think I have too little DNA, because I tend to loose it after the PCI step, even if I add acryl amide as carrier.

What would you think would happen if i just apply the upper aques phase from the back extraction method directly to the qiaamp micro column? Is that a crazy idea. People to it for the RNA extraction using the upper phase from the Trizol step and apply it directly to the RNeasy columns.

Best regards

Zeus
Reply With Quote
  #4  
Old 08-07-2009, 05:25 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Smile Re: Combining Trizol DNA extraction with QiaAMP micro purification

Your idea is perfectly fine.

Also note, if you are getting very little DNA, it's a good chance you have little contaminants and may not need the extra clean up steps like Phenol:chroloforms:IAA or qiamp column.
So I suggest you could also try using the First suspended DNA pellet after the back extraction buffer and isopropanol precipitation + EtOH wash.
Reply With Quote
Reply

Tags
combining , dna , extraction , micro , purification , qiaamp , trizol


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Trizol extraction - zebrafish embryos Kim Zebrafish Forum 0 11-04-2009 06:08 PM
Poor yield: DNA purification by phenol/chloroform extraction + ethanol precipitation? amesy Protocols and Methods Forum 1 03-27-2009 11:18 PM
Problem with extraction of DNA from Trizol max_rempel Epigenetics Forum: DNA Methylation, Histone and Chromatin Study 2 01-11-2008 03:39 PM
Extraction of protein using trizol IanMc Protocols and Methods Forum 1 10-27-2006 03:36 PM
Extraction of protein using trizol IanMc Protein Forum 1 10-27-2006 03:36 PM


All times are GMT. The time now is 06:56 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13131 seconds with 16 queries