Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing
Similar Threads
Thread Thread Starter Forum Replies Last Post
Low input/adaptor dimers whw Sample Prep / Library Generation 6 11-01-2011 11:26 AM
removal of primer dimers. shurjo Sample Prep / Library Generation 2 07-08-2011 01:12 PM
Avoiding primer dimers lpalacios 454 Pyrosequencing 9 05-05-2011 01:24 AM
PubMed: Extraction of high molecular weight DNA from microbial mats. Newsbot! Literature Watch 0 09-22-2010 02:00 AM
Primer Dimers in PEIn libraries Bruce E Illumina/Solexa 1 04-14-2010 05:46 PM

Thread Tools
Old 08-04-2009, 10:12 AM   #1
Junior Member
Location: Berkeley, CA

Join Date: Aug 2009
Posts: 2
Default Low Molecular Weight sample and primer dimers

I recently ran a Low Molecular Weight sample on our 454 and got a bad result. Most of the sequences were just primer dimers. Our samples are pooled PCR products, 200-600 bp in length. The LMW protocol says to skip the AMPure size selection/cleanup step after adapter ligation, but this allows primer dimers to be sequenced.
I want to eliminate the primer dimers, but I am afraid that if I do the AMPure cleanup, I will loose part of my sample DNA.
Any ideas about how to eliminate the primer dimers for the LMW sample?
I have attached a plot of the sequence read lengths from the last run.
Attached Images
File Type: jpg reads_length_TCAG_chart.jpg (13.3 KB, 27 views)
jsandler is offline   Reply With Quote
Old 08-26-2009, 01:27 AM   #2
Location: Germany

Join Date: Aug 2009
Posts: 33

Hi, if you don't want to use Ampure clean-up or gel extraction (I'm just assuming this would drop out for the same reasons), did you think about something like using a Centricon (or Microcon)? Millipore claims that they cannot only be used for proteins, but also provides DNA cut-off sizes:
Centricon-30 ssDNA cot-off: 60 nt, dsDNA cut-off: 50 bp
I think I have read something like 90% retention. I guess every additional step will not have 100% yield, but is that necessary? If your samples are PCR products, you should have plenty of everything.
Susanne is offline   Reply With Quote

adapter, ampure, low molecular weight, primer dimer, size selection

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 12:23 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2022, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO