Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
Primer dimer problem cybeline Illumina/Solexa 76 11-20-2018 11:00 AM
Two-step PCR protocol for Illumina sequencing EmmaUoW Illumina/Solexa 2 08-30-2013 12:08 AM
Primer dimer in ChIP-seq library lavagirl Sample Prep / Library Generation 7 06-07-2013 04:52 AM
primer dimer CCBIO 454 Pyrosequencing 5 03-18-2013 09:31 AM
PCR primer -- a PCR simulation on your computer gringer General 0 01-26-2012 02:03 AM

Thread Tools
Old 05-16-2014, 06:00 AM   #1
Location: Belgium

Join Date: Jan 2014
Posts: 21
Default primer dimer problem in PCR using Illumina protocol

I'm using the two step PCR Amplicon approach to to some targeted sequencing.
My PCR with my primers without adaptor sequences works just fine. But when I add adaptor sequences according to the protocol :
Append to 5 end of forward PCR primer:
Append to 5 end of reverse PCR primers:

all of a sudden, I only have primer dimers in my PCR product.
Anyone ran into this problem? How did you solve it?
Neiltje is offline   Reply With Quote
Old 05-16-2014, 03:11 PM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,236

This indicates that there is a primer heterodimer with strong bonds. First step would be to raise annealing temperature to favour your intended amplification while reducing unwanted primer-dimer interaction. If that is not helpful, next step would be to design new region specific primer(s) and analyse primer-primer interaction between your amplification primers (primers included with appended sequences) carefully to ensure that there is little interaction in your intended annealing temperature. You will need to take into account the salt concentration in your enzyme buffer as well.
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 12:35 AM.

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