SEQanswers

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



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
Amplicon sequencing - gel cut purification JKistler 454 Pyrosequencing 7 11-28-2012 11:14 AM
Fluidigm PCR amplicon Library prep yog77 Sample Prep / Library Generation 2 10-30-2012 04:36 AM
Library invisible on gel, despite Nanodrop results. Gel extraction problem? thdybwf Sample Prep / Library Generation 5 09-24-2012 04:41 AM
Library on gel but not on Bioanalyzer pauji Sample Prep / Library Generation 6 05-13-2012 05:30 PM

Reply
 
Thread Tools
Old 11-04-2012, 04:10 PM   #1
mbirnb
Junior Member
 
Location: CA, USA

Join Date: Nov 2012
Posts: 1
Default Disagreement between bioanalyzer and agarose gel for PCR amplicon library

Hello,

We are setting up for an illumina MiSeq run using a PCR amplicon-based library (adding the Illumina adaptors using nested PCR). Our results look quite clean via agarose gel (expected size ~310nt), but when our facility ran bioanalyzer we had quite variable results (ranging between 400 and 500nt, depending on the sample). There are seven samples, each with a different barcode, labeled 1-7.

I'm attaching both the gel and the bioanalyzer report - has anyone seen anything like this before?

Thanks,
Michael
Attached Images
File Type: jpg gel result.jpg (63.9 KB, 128 views)
Attached Files
File Type: pdf Bioanalyzer result.pdf (431.4 KB, 172 views)
mbirnb is offline   Reply With Quote
Old 11-05-2012, 03:47 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

I can only speculate. Maybe the agarose gel was run either without EtBr, then post run stained, or no EtBr was put in the gel or loading solution, but was put in the buffer? In either of these cases, the products would migrate mostly or entirely in the absence of EtBr.

Why is that important? Well, I have not tested this myself, but Dave Cook at Sage Science told me that in the absence of EtBr (or other DNA binding dyes) the difference in mobility between fully double-stranded amplicons, and "bubble products" (double stranded only at the adapter ends) does not happen. I.e. everything basically runs at the "correct" size.

So, if this is the issue you see, your amplicons are fine, but consist of variable amounts of normal and bubble-products. The Bioanalyzer chip faithfully displays the difference with the bubble products migrating slower than their true molecular weight.

Again, just a guess.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-13-2014, 01:47 PM   #3
DNA_Dan
Member
 
Location: Montana

Join Date: Nov 2008
Posts: 21
Default

Make sure you aren't overloading the chip. The first 3-5 samples look really hot relative to the standard. You could also try a DNA1000 chip since your products are so small.
DNA_Dan is offline   Reply With Quote
Old 07-30-2015, 03:45 PM   #4
tomhooven
Junior Member
 
Location: New York City

Join Date: Jul 2015
Posts: 1
Default

Hi Michael--
I was curious how this worked out for you. I recently prepared an amplicon library for TnSeq and had a similar discrepancy between my gel (the expected 190-bp band) and my bioanalyzer results (around 220 bp). I couldn't figure out why this happened.
-Tom
tomhooven is offline   Reply With Quote
Old 08-03-2015, 09:28 AM   #5
DNA_Dan
Member
 
Location: Montana

Join Date: Nov 2008
Posts: 21
Default

I think this has to do with the mobility of the y-forked adapters. Agarose is more difficult for migration because it's a solid phase, whereas the Bioanalyzer chip's gel matrix is much more fluid. I think the DNA 7500 chip gives the best representation of the "real" size.
DNA_Dan 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 06:29 PM.


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