SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Help needed regarding designing primers discoveradnan General 2 12-04-2015 08:34 PM
Designing primers on assembly: bizarre problem IanAWarren Illumina/Solexa 0 05-14-2015 02:57 AM
TruSeq DNA - need index/barcode-specific primers drops Illumina/Solexa 5 12-05-2012 12:24 PM
TruSeq PCR enrichment primers seangallaher Sample Prep / Library Generation 13 11-28-2012 01:42 AM
TruSeq compatible PCR primers ETHANol Illumina/Solexa 6 07-06-2011 06:35 AM

Reply
 
Thread Tools
Old 12-01-2016, 02:24 PM   #1
idedios
Member
 
Location: Irvine, CA

Join Date: Mar 2014
Posts: 18
Default Designing TruSeq LT Primers for IGH

Recently I created a primer design for immunoglobulin heavy chain rearrangement using Illumina TruSeq LT adapters and indexes. I'm using PCR primers from a multiplexed Sanger sequencing assay that my company currently uses and it works.

I'm somewhat uncertain where to attach the regions of interest to the primers as I plan on doing a single PCR. The typical designs for the adapters consist of a forked adapter however I didn't want to do separate hybridization and adapter ligation steps.

In my first design which amplified in a KAPA qPCR library quant but failed to generate clusters, I placed the Universal adapter upstream from the forward PCR primer sequence. The reverse-complement of the indexed adapter is also upstream of the reverse PCR primer sequence.

The adapter sequences I used were as follows:
Universal adapter (P5):
5-aat gat acg gcg acc acc gag atc tac act ctt tcc cta cac gac FORWARD-PCR-PRIMER-3
Indexed adapter (P7):
5-caa gca gaa gac ggc ata cga gat NNNNNN gtg act gga gtt cag acg tgt REVERSE-PCR-PRIMER-3

Is there something I'm missing here?

Last edited by idedios; 12-01-2016 at 02:31 PM.
idedios 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 10:22 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO