SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
The NEXTflex™ PCR-Free DNA Sequencing Kit Reduces Bias in NGS Library Prep Bioo Scientific Vendor Forum 10 12-20-2012 07:10 AM
coligation of PCR amplicon amhro Sample Prep / Library Generation 3 09-26-2012 05:12 AM
Access Array (platform from Fluidigm) for 454 library preparation Eric Normandeau 454 Pyrosequencing 4 09-02-2012 05:17 AM
PCR Jumping (Template Switching) in Amplicon sequencing eladSeq 454 Pyrosequencing 2 04-02-2012 07:10 AM
Library prep can you get both mRNA and small RNAs in the same library? willgordon Sample Prep / Library Generation 4 07-20-2011 11:31 AM

Reply
 
Thread Tools
Old 06-06-2012, 09:01 AM   #1
yog77
Member
 
Location: London

Join Date: Jun 2011
Posts: 18
Default Fluidigm PCR amplicon Library prep

Hi has any one got any advice on a two-step PCR amplicon library generation via barcode/adapter addition using a secondary PCR.

I am using the Fluidigm AA system have generated my Primary PCR amplicons 48 samples each with 48 amplocons and now need to attach the barcode/adapter via a secondary PCR. Fluidigm suggest using a 1in100 dilution of the primary PCR and adding the adapters using 15 cycles in a 20uL PCR.

My question is would it not be better to use a less dilute amount of the primary PCR say 1in20 or 1in40 dilution and cut the number of cycles to 12 or 13 and even in half the reaction volume (say 10uL), therefore reducing PCR errors by using fewer additional cycles? I just wondered if these modifications would be beneficial?

Thanks
yog77 is offline   Reply With Quote
Old 10-05-2012, 03:57 AM   #2
ugm6hr
Junior Member
 
Location: UK

Join Date: Feb 2011
Posts: 4
Default

Quote:
Originally Posted by yog77 View Post
Hi has any one got any advice on a two-step PCR amplicon library generation via barcode/adapter addition using a secondary PCR.

I am using the Fluidigm AA system have generated my Primary PCR amplicons 48 samples each with 48 amplocons and now need to attach the barcode/adapter via a secondary PCR. Fluidigm suggest using a 1in100 dilution of the primary PCR and adding the adapters using 15 cycles in a 20uL PCR.

My question is would it not be better to use a less dilute amount of the primary PCR say 1in20 or 1in40 dilution and cut the number of cycles to 12 or 13 and even in half the reaction volume (say 10uL), therefore reducing PCR errors by using fewer additional cycles? I just wondered if these modifications would be beneficial?

Thanks
This makes perfect sense, but I didn't want to deviate from their published protocols for my experiment.
If you do use an alternate dilution / number of PCR cycles, I'd be interested to hear the outcome.
ugm6hr is offline   Reply With Quote
Old 10-30-2012, 04:36 AM   #3
yog77
Member
 
Location: London

Join Date: Jun 2011
Posts: 18
Default

After trying a few different dilutions and cycle combinations and checking them on the bioanalyzer. I went with the following:

1uL of the primary PCR product as template for a 5uL secondary/barcoding PCR with only 8 cycles. This worked very well and gave good sequencing results. If you require further details let me know.
yog77 is offline   Reply With Quote
Reply

Tags
library generation, library prep, pcr sequencing

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 12:19 PM.


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