SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
454 titanium emulsion PCR problems? kjones 454 Pyrosequencing 21 03-21-2012 08:46 AM
EZ bead Bubbles after transfering emulsion asaleh SOLiD 1 06-20-2011 04:32 AM
one bead-one fragment? jean_valjean General 3 12-21-2010 02:27 PM
Emulsion PCR for SOLiD Sequencing Kanak Vaidya General 0 12-07-2010 02:45 AM
SOLID emulsion PCR using other than the ABI9700 kwaraska SOLiD 1 01-21-2009 07:52 PM

Reply
 
Thread Tools
Old 11-30-2009, 01:21 AM   #1
Triticum
Member
 
Location: Austria

Join Date: Sep 2009
Posts: 12
Default emulsion pcr - one template-fragment per bead?

hi!
how is it assured, that only one fragment per bead is contained in an emulsion-droplet? why canīt there be more different(!) template-fragemts per bead, which would lead to nonsense-sequences?

triticum
Triticum is offline   Reply With Quote
Old 11-30-2009, 10:31 AM   #2
454Sequencing
Member
 
Location: Branford, CT

Join Date: Apr 2009
Posts: 22
Default

Hi Triticum,

In fact, there is a certain percentage of droplets that contain more than one template fragment. We minimize the occurrence of this via appropriate library dilution. A good reason for functional characterization (i.e. enrichment titration) is that you can ascertain the percent enrichment at a given copy-per-bead and thereby have some expectations about the quality of sequencing expected when you scale up the emPCR reactions. Very high enrichment could be an indicator that your library copy-per-bead is too high.

In any case, PicoTiterPlate wells containing beads with multiple fragments will not yield productive sequence and will be removed as Mixed Reads during quality filtering.

Best regards,
Jason

Quote:
Originally Posted by Triticum View Post
hi!
how is it assured, that only one fragment per bead is contained in an emulsion-droplet? why canīt there be more different(!) template-fragemts per bead, which would lead to nonsense-sequences?

triticum
__________________
Technical Product Manager
454 Life Sciences, A Roche Company
454Sequencing 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 06:43 PM.


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