SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Agilent automated library preps-Low yields after pre-hybridisation PCR debbiehughes Sample Prep / Library Generation 0 11-21-2011 06:06 AM
Library PCR enrichment questions (with image!) ZAAB Sample Prep / Library Generation 6 06-07-2011 06:47 PM
massive multiplex PCR-library for 454-sequencing CG&R Sample Prep / Library Generation 3 02-10-2011 01:00 AM
Small RNA sequencing library: Help! Very Strange PCR products MarineMan Illumina/Solexa 1 12-23-2010 04:31 AM
SPRI bead for PCR cleanup in illumina pair end library preparation Berkeley2010 Illumina/Solexa 2 10-20-2010 12:46 PM

Reply
 
Thread Tools
Old 04-19-2010, 05:43 PM   #1
yuan.li
Junior Member
 
Location: CA

Join Date: Oct 2009
Posts: 2
Question Library Multimerizing After PCR

This is the most weird thing. I wonder if anyone has seen this problem:
On Agilent DNA 1000 chip, my paired end library after PCR amplification has a little bit of high molecular weigh stuff that looks like multimers of the fragment of interest (~300bp). So I purified it to remove the high MW stuff and re-ran it on bioanalyzer but again I saw that same multimer!

Any idea what it is? I thought it was a PCR artifact initially. If that was the case it should be gone after the purification step but it's not. Is it a real multimer?
yuan.li is offline   Reply With Quote
Old 04-20-2010, 03:43 AM   #2
sulfobus
Member
 
Location: Denmark

Join Date: Oct 2009
Posts: 12
Default

It could be heteroduplexes forming during late stage PCR or during purification (esp. using chaotropic detergents s.a. guanidine salts in combination with heat). They migrate slower on gel, check section II in Kanagawa T (2003).

Edit: This should only be prominent if you have homologous regions in your sequences. But maybe the adapter regions are long enough to bring non-identical DNA strands together.

Last edited by sulfobus; 04-20-2010 at 03:50 AM.
sulfobus 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 06:19 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO