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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Disagreement between bioanalyzer and agarose gel for PCR amplicon library mbirnb Sample Prep / Library Generation 4 08-03-2015 09:28 AM
Combine TruSeq DNA PCR-Free library and 2x300bp PE sequencing on MiSeq Johnny99 Sample Prep / Library Generation 4 08-03-2015 12:57 AM
'Assembly PCR' for homemade amplicon library prep esherman Illumina/Solexa 18 11-22-2014 03:28 PM
PCR step in DNA library prep? liumangmang Illumina/Solexa 5 10-09-2014 02:16 PM
Fluidigm PCR amplicon Library prep yog77 Sample Prep / Library Generation 2 10-30-2012 04:36 AM

Thread Tools
Old 11-07-2019, 09:37 AM   #1
Senior Member
Location: New England

Join Date: Jun 2012
Posts: 200
Default PCR amplicon library - DNA smearing

I've been making two-step amplicon libraries for years now with very few problems. The original amplicons are tailed with partial Nextera xt adapters and then I use Phusion HF to add the rest of the adapter with 6 cycles of PCR and KAPA Pure beads for cleanup. Recently, the yield has been much lower than expected and, rather than a pretty clean peak on the Bioanalyzer, there's been a lot of high molecular weight smearing. The samples labeled SPS01-07 are after the second round of amplication while the samples labeled Pre are before. Suggestions as to what might be going on?
Screen Shot 2019-11-07 at 1.34.30 PM.png
microgirl123 is offline   Reply With Quote
Old 11-07-2019, 04:24 PM   #2
Senior Member
Location: US

Join Date: Dec 2010
Posts: 458

Not a good explanation, nevertheless: perhaps your stock of index primers is degrading or got contaminated? You might want to order a few fresh oligos and compare?
luc is offline   Reply With Quote
Old 01-31-2020, 01:31 PM   #3
Junior Member
Location: Fort Collins, CO

Join Date: Jan 2020
Posts: 2

Hey there, microgirl:

I was wondering if you tried what luc suggested and if this worked.

Also, do you have a low annealing temperature on your PCR cycles? I figure it's stayed the same, but since other things have been changed, maybe the thermocycler programming needs to be altered as well.

I've only been doing library prep for around 1.5 yr, but it surprised me how low the annealing temps in a lot of protocols are set to. We anneal at 50C!
aesterle 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 08:33 PM.

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