SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Gel-Purify, columns or silica matrix? scuellar Illumina/Solexa 6 02-07-2017 05:55 AM
Results with mate-pair preps dzerbino Illumina/Solexa 8 05-21-2011 12:02 PM
Interpretation of columns in MAQ SNP output? griffon42 Bioinformatics 3 09-13-2010 01:36 AM
SPRI-TE Illumina library preps GW_OK Sample Prep / Library Generation 5 08-23-2010 09:13 AM
qiagen and minelute columns seqgirl123 Illumina/Solexa 0 03-09-2009 04:59 AM

Reply
 
Thread Tools
Old 06-09-2011, 12:35 PM   #1
bbeitzel
Member
 
Location: Ft. Detrick, MD

Join Date: Aug 2008
Posts: 50
Default MinElute columns for TruSeq preps?

Is there any reason why you can't use MinElute columns instead of Ampure beads during a TruSeq DNA library prep? I can't see any reason for using beads instead of columns after the end repair and A tailing reactions. I can see maybe using beads after adapter ligation to remove adapter dimers, but couldn't you accomplish the same thing by using a MinElute purification followed by gel purification to remove dimers? I know that if you have a lot of adapter dimers that some of those may contaminate larger gel fractions, but is that contamination much of a problem during the limited cycle enrichment PCR?

I am new to TruSeq library preps, so I am trying to understand the reliance on Ampure beads when MinElute purifications are so much faster (compared to the bead steps in the TruSeq protocol.)

Thanks for any thoughts/info.
bbeitzel is offline   Reply With Quote
Old 06-10-2011, 12:25 AM   #2
protist
Senior Member
 
Location: Ireland

Join Date: Jan 2009
Posts: 101
Default Re MinElute columns for TruSeq preps?

There should not be any reason not to. Using the beads for each reaction allows automation thus a more high-throughput libaray generation process.
If you look at the TruSeq DNA Sample Prep kit info you will see for gDNA libraries they still recommend gel isolation followed by a MinElute clean-up - for the TruSeq RNA libraries all clean-ups or size selections are bead based.
protist is offline   Reply With Quote
Old 05-03-2012, 09:36 AM   #3
lterhune
Member
 
Location: Boulder, CO

Join Date: Sep 2011
Posts: 19
Default

On a related note-- does anyone know why there is a double purification with AMPure beads after the ligation? This seems unnecessary at first glance, especially when you are gel purifying at the next step.
lterhune is offline   Reply With Quote
Old 05-03-2012, 09:43 AM   #4
protist
Senior Member
 
Location: Ireland

Join Date: Jan 2009
Posts: 101
Default

Quote:
Originally Posted by lterhune View Post
On a related note-- does anyone know why there is a double purification with AMPure beads after the ligation? This seems unnecessary at first glance, especially when you are gel purifying at the next step.
For the TruSeq RNA Sample Prep Kits you don't do gel isolations, all clean-up steps are bead based.
protist is offline   Reply With Quote
Old 05-03-2012, 09:51 AM   #5
lterhune
Member
 
Location: Boulder, CO

Join Date: Sep 2011
Posts: 19
Default

@protist: Sorry for not clarifying-- I was talking about the TruSeq DNA sample prep kit. We always do the gel option, not gel-free.
lterhune is offline   Reply With Quote
Old 05-03-2012, 10:22 AM   #6
greenhilly
Member
 
Location: RTP, NC

Join Date: Jan 2012
Posts: 11
Default

Quote:
Originally Posted by bbeitzel View Post
Is there any reason why you can't use MinElute columns instead of Ampure beads during a TruSeq DNA library prep? I can't see any reason for using beads instead of columns after the end repair and A tailing reactions. I can see maybe using beads after adapter ligation to remove adapter dimers, but couldn't you accomplish the same thing by using a MinElute purification followed by gel purification to remove dimers? I know that if you have a lot of adapter dimers that some of those may contaminate larger gel fractions, but is that contamination much of a problem during the limited cycle enrichment PCR?

I am new to TruSeq library preps, so I am trying to understand the reliance on Ampure beads when MinElute purifications are so much faster (compared to the bead steps in the TruSeq protocol.)

Thanks for any thoughts/info.
Had the same question. If you try the minElute in lieu of AmpPure beads, please let us know how it goes.
greenhilly is offline   Reply With Quote
Old 05-03-2012, 10:47 AM   #7
protist
Senior Member
 
Location: Ireland

Join Date: Jan 2009
Posts: 101
Default

Quote:
Originally Posted by lterhune View Post
@protist: Sorry for not clarifying-- I was talking about the TruSeq DNA sample prep kit. We always do the gel option, not gel-free.
My bad assuming it was RNAseq. I still do the gel selection for all gDNA libraries and MinElute prior to the gel isolation step. There should not be a problem and I agree with the gel option there should be no requirement for a double bead selection.
protist is offline   Reply With Quote
Old 05-03-2012, 12:00 PM   #8
Veleno
Member
 
Location: UK

Join Date: Aug 2011
Posts: 16
Default

Less loss of material.
Veleno is offline   Reply With Quote
Old 05-06-2012, 11:34 PM   #9
riehle
Junior Member
 
Location: Freiburg

Join Date: Apr 2012
Posts: 7
Default

Hello...
I came across the same question that you were posting last year: Can the Ampure Step in the Illumina DNA Sample prep protocol be replaced by MinElute Columns?
Have you tried this out by now? Would you use for elution of the DNA from the columns the volumes indicated in the Illumina protocol or is there some adjustment needed?
riehle is offline   Reply With Quote
Old 05-10-2012, 07:49 AM   #10
RNAseqer
Member
 
Location: London

Join Date: Sep 2010
Posts: 22
Default

You can't efficiently remove the longer style adapters with columns. That's why beads are required.
RNAseqer is offline   Reply With Quote
Old 05-11-2012, 04:46 AM   #11
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Also if you have enough samples to process in a 96-well plate with a multi-channel pippetter and a good plate magnet, then AMPure is, by far, less time consuming than working with individual tubes.

--
Phillip
pmiguel is offline   Reply With Quote
Reply

Thread Tools

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




All times are GMT -8. The time now is 02:02 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO