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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Ampure versus Ampure XP seqgirl123 Sample Prep / Library Generation 49 03-19-2015 04:02 AM
Bizarre RNAse cleanup problem HereBeDragons Sample Prep / Library Generation 1 07-24-2013 05:18 AM
low yield of SOLiD templated beads KevinLam SOLiD 1 10-06-2010 03:24 PM
RNA cleanup after fragmentation - illumina Paulo.jt RNA Sequencing 2 07-02-2010 05:00 AM
Wiki cleanup? krobison Wiki Discussion 3 05-02-2010 03:12 PM

Thread Tools
Old 08-21-2012, 09:34 AM   #1
Junior Member
Location: Maritime Canada

Join Date: Apr 2012
Posts: 5
Default Low yield with Ampure XP cleanup

Hi all,
I am having some issues with my Ampure cleanup step and would love some troubleshooting help.

I'd say that every time I use the ampure beads (at 1.8 ratio) I lose about 50% of my product, which seems like A LOT. (I've tried my best to optimize the pcr conditions, but I think the biggest problem is that my template DNA is super crappy (literally - it is extracted from feces) and full of inhibitors.)

I'm already struggling to get enough ng of product made for NGS, so I'm pretty tired of losing it (and this high quality taq is expensive!) The beads expire in 2013. I make new EtOH every time. Might anyone have any idea about what could be going wrong??
akbowser is offline   Reply With Quote
Old 08-21-2012, 09:53 AM   #2
Location: Ireland

Join Date: Sep 2011
Posts: 86

That's quite a lot of loss for one single-sided Ampure cleanup. Bear in mind, though, that Ampure XP cleanup will eliminate DNA under a certain size, so is it possible that the DNA loss you're measuring is just primers/dNTPs/small fragments being removed from the mix? What method did you use to quantify your DNA pre- and post-cleanup?
Rocketknight is offline   Reply With Quote
Old 08-21-2012, 09:55 AM   #3
Senior Member
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317

What is your assay method for determining your mass of DNA at each step? Could you give us a particular example? That is pick a typical sample and go through the yield you have at each step. (Make sure to give us total mass recovered at that step, not just the concentration.)

If you are initially assaying the amount of DNA using UV spectrophotometry, it is likely you are overestimating the amount of DNA you have. Generally >50% (and often >90%) of the absorbance at 260 nm of the typical genomic DNA prep derives from stuff that is not DNA. (RNA, degraded RNA, phenol, etc.)

pmiguel is offline   Reply With Quote
Old 08-22-2012, 12:46 PM   #4
Senior Member
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119

The protocol says that overdrying the beads can significantly decrease yield, have you tried using a shorter drying time?
kerplunk412 is offline   Reply With Quote
Old 08-22-2012, 02:08 PM   #5
Location: Texas

Join Date: Jul 2012
Posts: 26

What about the expiration date? I am generally curious if people have any experience with "expired" Ampure XP beads? I have been inadvertently using some which are just a couple of months out of date and have ordered fresh, but I'm wondering what the real shelf life is, as they seem to still be performing fairly well.
docbio is offline   Reply With Quote
Old 08-27-2012, 06:08 AM   #6
Location: USA

Join Date: Nov 2011
Posts: 17
Default underdrying beads

Not getting all of the ethanol off the beads can also significantly reduce yields. make sure that you see small cracks and that the color lightens up. I have not had a problem with overdrying.
SarahNGS is offline   Reply With Quote

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 02:51 PM.

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