Go Back   SEQanswers > General

Similar Threads
Thread Thread Starter Forum Replies Last Post
Non-specific band after gel purification of PCR products omnivore Sample Prep / Library Generation 3 04-05-2017 06:19 PM
Agencourt RNAclean XP bead purification troubleshooting Terazax Sample Prep / Library Generation 2 07-30-2015 07:00 AM
Problem with AMPure purification JanaSEQ Sample Prep / Library Generation 11 09-22-2014 03:09 PM
Ampure XP beads Dual Bead-based Size Selection ratio and PCR purification Leo Lee Sample Prep / Library Generation 4 09-19-2014 09:36 AM
Agencourt Ampure purification kit - exp date ntremblay Sample Prep / Library Generation 3 03-06-2012 03:41 AM

Thread Tools
Old 04-02-2016, 03:45 PM   #1
Junior Member
Location: zurich

Join Date: Apr 2016
Posts: 1
Default Different band size before and after purification with Agencourt AMPure

Hi everyone,

I'm doing amplification and purification of 4 different genomic regions. 3 of them are ok, but I'm struggling with my last amplicon. The amplicon length on the gel changes before and after the purification. Before purifying I have a 360 bp and after the purification I have a strong lower band of 250 and a upper band around 400 pb. It cannot be caused by a higher DNA yeld before the purification because I loaded different and small volumes on the gel to be sure about the size. It happened the same with a different primer set for the same region. Thinking about an issue with my primer set I changed it, but the problem reappeared.

Is it possible that somehow the beads interfere with the size of the amplicon? This problem never happened with other genes so I really don't understand what's going on.

Thank you for any suggestion!

ElenaCH is offline   Reply With Quote
Old 04-02-2016, 07:49 PM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238

Posting gel photo can give some clues on the issue. Purification cannot change the size of DNA fragment but it can cut some small fragments depending on bead/amplicon solution ratio. Amplicon before purification would be in PCR buffer with different salt concentration than after that can affect DNA migration speed affecting sizing. It is also possible that some DNA is denaturing during purification which would run as shadow band on the gel. To further investigate running on different gel%, voltage, diluting PCR before run, loading equal amplicon mass before and after purification would be useful.
nucacidhunter 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 10:21 AM.

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