SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
ampure beads sc10021 Sample Prep / Library Generation 12 07-03-2017 01:26 AM
Different band size before and after purification with Agencourt AMPure ElenaCH General 1 04-02-2016 07:49 PM
Illumina Sample Purification Beads vs AMPure XP Beads JER Illumina/Solexa 2 03-29-2015 01:36 AM
Ampure XP beads Etherella Illumina/Solexa 4 10-16-2013 05:05 AM
Agencourt Ampure purification kit - exp date ntremblay Sample Prep / Library Generation 3 03-06-2012 03:41 AM

Reply
 
Thread Tools
Old 10-13-2016, 04:02 AM   #1
Sylv
Junior Member
 
Location: Norway

Join Date: Oct 2016
Posts: 6
Default Agencourt Ampure XP beads

Hi.
I am testing some DNA samples for doing ddRAD sequencing, and I have a problem.
After restriction digest, I purify the reaction using Agencourt Ampure XP beads. When eluting the DNA in water, the beads are stuck to the wells, and very difficult to resuspend.

Have anyone experienced the same problem, and have som ideas as to what causes this? I was thinking the beads have been drying to long, but drying them for shorter time did not make any difference. A pH problem should affect binding of DNA to the beads, and not stick the beads to the wells right??

I hope someone have some advice for me
Sylv is offline   Reply With Quote
Old 10-15-2016, 01:26 PM   #2
heusssss
Junior Member
 
Location: Boston

Join Date: Feb 2014
Posts: 1
Default

Including 0.05% (v/v) Tween-20 in your eluent will prevent bead clumping (and has a bonus effect of preventing DNA adsorption of surfaces in general, which can be a problem when you are working with very low concentrations of DNA). I use a low concentration of tween in virtually all of my buffers (except for the 80% ethanol I use to wash ampure beads).

Another thing that can cause bead clumping is a high amount of protein, cell debris, or other contaminating substances in the original sample. I've generally found that this won't hurt DNA yield. Sometimes beads might be so chunky that a lot of pipetting up and down, vortexing, incubation on the bench, then more vortexing is required to get them (mostly) resuspended. But even in this case I seem to have gotten acceptable yields of purified DNA.

Last edited by heusssss; 10-15-2016 at 01:29 PM. Reason: added more info
heusssss is offline   Reply With Quote
Old 10-17-2016, 01:20 AM   #3
Sylv
Junior Member
 
Location: Norway

Join Date: Oct 2016
Posts: 6
Default

Quote:
Originally Posted by heusssss View Post
Including 0.05% (v/v) Tween-20 in your eluent will prevent bead clumping (and has a bonus effect of preventing DNA adsorption of surfaces in general, which can be a problem when you are working with very low concentrations of DNA). I use a low concentration of tween in virtually all of my buffers (except for the 80% ethanol I use to wash ampure beads).

Another thing that can cause bead clumping is a high amount of protein, cell debris, or other contaminating substances in the original sample. I've generally found that this won't hurt DNA yield. Sometimes beads might be so chunky that a lot of pipetting up and down, vortexing, incubation on the bench, then more vortexing is required to get them (mostly) resuspended. But even in this case I seem to have gotten acceptable yields of purified DNA.
Thank you for your reply. The beads does not clump together as such when adding them to the DNA, they just stick to the wells of the plate. The quality of the DNA was checked using the NanoDrop ratios, and they are just fine. So there should not be any contamination of proteins or other things in the samples.
Will try using Tween in the eluting agent..thank you.
Sylv is offline   Reply With Quote
Old 10-17-2016, 09:18 AM   #4
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 415
Default

Hi Sylv,

perhaps your are drying your beads too long after the 80% EtOH washes.
luc is offline   Reply With Quote
Old 10-17-2016, 10:04 AM   #5
cmbetts
Senior Member
 
Location: Bay Area

Join Date: Jun 2012
Posts: 109
Default

I would venture to guess that it's probably got something to do with the buffer used in the digest. Several years ago, I was screening polymerases for library amplification and found that the different buffers had a huge effect on the bead pelleting characteristics. Generally, the best bet was to spike some detergent into the reaction immediately before adding the beads. Alternatively, going to a beefier magnet can also make a big difference.
cmbetts is offline   Reply With Quote
Old 10-17-2016, 10:37 PM   #6
Sylv
Junior Member
 
Location: Norway

Join Date: Oct 2016
Posts: 6
Default

Quote:
Originally Posted by luc View Post
Hi Sylv,

perhaps your are drying your beads too long after the 80% EtOH washes.
Hi, and thanks for you input.
I also thought of this, as Illumina protocol has very long drying of the beads. But I tried drying them for a shorter time, and it made no difference.
Sylv is offline   Reply With Quote
Old 10-17-2016, 10:44 PM   #7
Sylv
Junior Member
 
Location: Norway

Join Date: Oct 2016
Posts: 6
Default

Quote:
Originally Posted by cmbetts View Post
I would venture to guess that it's probably got something to do with the buffer used in the digest. Several years ago, I was screening polymerases for library amplification and found that the different buffers had a huge effect on the bead pelleting characteristics. Generally, the best bet was to spike some detergent into the reaction immediately before adding the beads. Alternatively, going to a beefier magnet can also make a big difference.
HI, and thanks for you input.
I thought of that, but the same buffer has been used several times perviously without causing trouble. It was also done the same procedure on DNA samples isolated by two different methods, and the one isolated using the Qiagen DNeasy kit caused the problem. Samples isolated using the CTAB method caused no problems. The magnet is no problem, the problem is that the beads stick to the wells.
Sylv 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 02:27 PM.


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