Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing

Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq: TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Newsbot! Literature Watch 5 07-13-2013 01:02 AM
PubMed: Barcoded primers used in multiplex amplicon pyrosequencing bias amplification Newsbot! Literature Watch 0 09-06-2011 03:00 AM
tophat fusion --fusion-min-dist MerFer Bioinformatics 1 07-24-2011 08:09 PM
HPLC purification of 454 fusion primers skolyar 454 Pyrosequencing 2 09-22-2010 01:40 PM
Poor v.3 run westerman SOLiD 10 03-16-2010 08:01 AM

Thread Tools
Old 12-09-2010, 04:04 AM   #1
Location: EU

Join Date: Sep 2010
Posts: 24
Default poor amplification with fusion primers


we're having trouble with some of our barcoded 16s primers. We are using 27f(fused with primer B) and 519r (fused with MID and primer A). These exact primers have been used before by others (Wu et al 2010) with much success.

We have 16 different barcoded reverse primers (with MIDs 1,2,3,4,5,6,7,8,10,11,13,14,15,16,17,18) and use the same forward primer for each. Unfortunately, primers with MIDs 1,2,7,8&16 do not amplify and MIDS 5,14 & 15 amplify very poorly. At first I thought it was a problem with the cognate samples rather than the barcoded primers but the samples yield full-length 16S amplicons with 27f (fusion primer) and 1492r. Even in the same PCR run as the 27f/519r. Also the dodgy MIDs are equally rubbish at amplifying E. coli DNA. Does anyone have experience of dodgy barcoded primers? They were pretty expensive...

Seqasaurus is offline   Reply With Quote
Old 12-11-2010, 12:21 PM   #2
Location: London

Join Date: Jun 2010
Posts: 27


Would you please provide me with a link to the paper (Wu et al 2010) you are referring to? I would like to read the experimental part and then see whether I am experiencing the same problem you are describing here.

Xterra is offline   Reply With Quote
Old 05-04-2013, 10:50 AM   #3
Junior Member
Location: Hong Kong

Join Date: May 2013
Posts: 1

i also met the same problem and i got 40 MIDs fusion primer for amplification. ANyone got the idea to solve the problems?
mydogphy is offline   Reply With Quote
Old 05-23-2013, 07:20 AM   #4
Junior Member
Location: Prague

Join Date: Jul 2012
Posts: 1

We have used many fusion primers both for 16s and functional genes. The majority of problems were actually connected to type of polymerase rather than the primers themselfs. So now we do sort of primer-polymerase optimalization.

Common problems are either no products or multipleproducts. We also experienced contamination in brand new polymerase with DNA, probably originating from a production strain. Lately these problems ocur more often than 2 years ago.
Strejda 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 04:30 AM.

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