SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
454 GS Junior for sale (complete system) pfeifferr 454 Pyrosequencing 0 12-13-2012 01:07 PM
454 junior user - hello everyone Encarnita Introductions 0 07-18-2012 01:26 AM
free 454 junior kits mehtamorphosis 454 Pyrosequencing 0 04-17-2012 04:48 PM
454 Junior MIDs newendophytologist 454 Pyrosequencing 2 02-24-2012 01:34 PM
ssaha2 with 454 Junior jtrivino Bioinformatics 0 05-13-2011 09:22 AM

Reply
 
Thread Tools
Old 02-06-2013, 05:32 AM   #1
Pascale
Junior Member
 
Location: France

Join Date: Feb 2013
Posts: 3
Default Metagenomic with 454 Junior

I try to sequence multiplex (21 samples) amplicons for months !! Amplicon size is around 609 pb (with MID ...). The problem is that the yield is very low (2%) with 2 or 4 cpb and when I sequence I juste have 50 bp instead of 609 pb (bioanalyzer is ok, no small fragments).

Do you think to increase elongation time of emPCR will be better or the problem come from an other step ?

Thank for your help !
Pascale is offline   Reply With Quote
Old 02-06-2013, 12:22 PM   #2
Aniki
Member
 
Location: Toronto

Join Date: Mar 2011
Posts: 39
Default

Quote:
Originally Posted by Pascale View Post
I try to sequence multiplex (21 samples) amplicons for months !! Amplicon size is around 609 pb (with MID ...). The problem is that the yield is very low (2%) with 2 or 4 cpb and when I sequence I juste have 50 bp instead of 609 pb (bioanalyzer is ok, no small fragments).

Do you think to increase elongation time of emPCR will be better or the problem come from an other step ?

Thank for your help !
You are experiencing things that a lot of users are experiencing. Do the following to minimize your losses:

1) increase your cpb to between 15 and 30cpb. I know this seems insane but it's the only way.
2) get a KAPA qPCR Lib-A/Lib-L kit to accurately quantify libraries.
3) ask for newest lots to be shipped to you and hope.
Aniki is offline   Reply With Quote
Old 02-06-2013, 04:51 PM   #3
RCJK
Senior Member
 
Location: Australia

Join Date: May 2009
Posts: 155
Default

You can also try the long amplicon emPCR protocol since yours is >600bp.
RCJK is offline   Reply With Quote
Old 02-07-2013, 01:11 AM   #4
Pascale
Junior Member
 
Location: France

Join Date: Feb 2013
Posts: 3
Default

Thank you very much for your advice ! I will try all of that this afternoom and I will let you know. Have a good day.
Pascale is offline   Reply With Quote
Old 03-14-2013, 05:52 AM   #5
Palecomic
Member
 
Location: London

Join Date: Aug 2010
Posts: 34
Default

We size select twice with Ampure XP for amplicon sequencing, primer (and/or primer dimer) that is undetectable by bioanalyzer can still lead to a big short read spike. Our amplicon is 676 bp and the long emPCR protocol had an instant and amazing effect on improving the runs on our Jr.

Our optimized cbp is 0.35 - so seems very variable from site to site. The kapa kits are expensive, particularly relative to a Jr run, so we don't use them.
Palecomic 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 11:51 PM.


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