SEQanswers

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
Ampure XP buffer cli Sample Prep / Library Generation 8 12-16-2014 10:26 PM
Ampure XP inconsistency HMorrison Sample Prep / Library Generation 6 11-03-2011 11:58 AM
Innovative Solutions vs AMPure suefo 454 Pyrosequencing 2 10-14-2011 06:22 PM
Ampure XP automation HGENETIC Sample Prep / Library Generation 0 07-19-2010 08:24 AM

Reply
 
Thread Tools
Old 11-29-2010, 04:34 PM   #1
sjcire
Junior Member
 
Location: Corvallis OR

Join Date: Nov 2010
Posts: 4
Default Ampure XP Hack

For Chip-Seq library generation, in the size select step, I have had many problems using agarose; low resolution of short nucleotides and low % recovery using the min-elute columns. I prefer to use a TBE acrylamide gel. In order to do so, acrylamide gels electro-elution was employed, see Sambrook's molecular cloning. The problem with eletro-eluiton, in my hands, is that in the end I'm left with > 350ul of a dilute DNA solution. To use with Ampure beads, following the standard protocol, > 630ul of Ampure was required. This makes it an expensive step. I therefore have made an attempt to hack the kit. Please find attached the protocol I use and its companion excel spreadsheet. Archive.zip

I have used this protocol to purify 1ml 1ng/ul DNA sample with 10ul Ampure beads. I achieved ~40% recovery. This may save you 1 EtOH precipitation or save some of your Ampure XP.
I should add that standard column purification is easier and better suited for this type of application. However, Ampure XP has better size exclusion properties(removal of primers and adapter dimers). These properties make it well suited for ChIP-Seq library generation, in my opinion.

Please let me know about typos and mistakes.

Last edited by sjcire; 11-29-2010 at 04:38 PM.
sjcire is offline   Reply With Quote
Old 07-02-2012, 07:16 AM   #2
mikmz
Junior Member
 
Location: Ireland

Join Date: Sep 2011
Posts: 2
Default

Thank you for this information, it was very helpful.

Do you know if the Ampure beads are silica-coated and functionalised with -COOH? If so, is the buffer that you have described a -COOH-specific buffer?

I have heard that the patent for this buffer has run out.

Also, can you explain how you came to calculate the correct volumes of PEG and NaCl in the buffer, so that the correct final concentration of each is reached?

Thanks.

Last edited by mikmz; 07-03-2012 at 01:13 AM.
mikmz 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 11:28 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO