SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Switching file formats Kezzie General 2 03-14-2014 11:41 AM
Slow Pileup After Switching to Stampy groundcover Bioinformatics 4 04-11-2012 07:28 AM
Can you really see miRNAs on gels before PCR amplification fisher Illumina/Solexa 3 07-27-2010 06:37 PM
4% agarose gels? ik76 Sample Prep / Library Generation 1 04-01-2010 10:39 AM
are Novex TBE PAGE gels really necessary for DGE? jasonc Illumina/Solexa 0 09-07-2009 09:01 AM

Reply
 
Thread Tools
Old 10-23-2014, 10:55 AM   #1
docphil
Junior Member
 
Location: Seattle WA

Join Date: Aug 2014
Posts: 8
Default Ugly gels after switching to 384w plate from 96

Hello,
My lab use's custom primer sets for HLA genotyping using the Illumina MiSEQ. We have optimized our multiplex primer sets for initial amplification using 96 well PCR plates. These reactions produce clean, cookie cutter bands when ran on 1.5% TBE gels. We have recently been experimenting with 384well plates and have found that when using the same reagent concentration, rxn volume (20ul) and thermal cycling conditions, the gels from the 384well rxns have non-specific bands and bad primer-dimers. I have tried lowering rxn volume to 10ul while maintaining original reagent proportions, varying mg2+ concentration, and increasing extension time all with unconvincing results. The 384well peltier blocks are calibrated and well maintained so it is confusing to me why we cant just scale up our procedure. Any one have some experience with switching to 384 plates? Some advice would be greatly appreciated. Thanks!
docphil is offline   Reply With Quote
Reply

Tags
384, miseq, multiplex, non-specific band, primer dimer

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 03:11 AM.


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