SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
KAPA mRNA-seq kit, would you recommend it? N311V Sample Prep / Library Generation 7 11-17-2015 05:35 AM
MiSeq -- Kapa Library Kit for Clostridia? jhalpin Sample Prep / Library Generation 0 03-05-2015 11:46 AM
Kapa library quantification kit seqgirl123 Sample Prep / Library Generation 1 11-10-2014 12:14 PM
Illumina sequences with Kapa kit seqgirl123 Sample Prep / Library Generation 1 03-29-2013 09:42 AM
KAPA qPCR kit for XL+ libraries? madseq 454 Pyrosequencing 2 08-31-2012 07:39 AM

Reply
 
Thread Tools
Old 09-23-2020, 05:16 PM   #1
rougaroux
Junior Member
 
Location: NYC

Join Date: Sep 2020
Posts: 3
Default No amplification with Kapa kit

Hello,
I have been using the Kapa Hyper Prep kit to try and prep DNA libraries for target enrichment.
I am using custom Y adapters and dual indexed primers which I know have been used successfully with the Kapa kit before.
My issue is that I am getting virtually no amplification from the PCR. By increasing the number of cycles up to 16, my library concentrations increased by only 2-3X.


The kit I'm working with is new.
I checked my shearing with the bioanalyzer and all samples were within the targeted range (400-600 bp).

I suspect the problem is with adapter ligation...after PCR, I see very high concentrations with a nanodrop (but not Qubit) that went down sharply after bead cleanup. This makes me think lots of adapter dimers and/or unused primers were present.

Has anyone here experienced similar issues with the Kapa kit, or does anyone have any advice on how best to troubleshoot this problem?
rougaroux is offline   Reply With Quote
Old 09-26-2020, 06:39 AM   #2
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 451
Default

You could run aliquots of the reaction (before and after bead cleanups) on an agarose gel to see what is going on.
If the ligation does not work porperly, perhaps your samples have some chemical contamination?
luc is offline   Reply With Quote
Old 09-30-2020, 11:21 AM   #3
rougaroux
Junior Member
 
Location: NYC

Join Date: Sep 2020
Posts: 3
Default

Thanks for the suggestion, Luc.
Do you know if I would be able to reliably identify unincorporated adapters on a gel?
rougaroux is offline   Reply With Quote
Old 10-01-2020, 09:57 AM   #4
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 451
Default

Depends on the dye used and the amount of primers. Ethidiumbrmide is ten times less sensitive for ssDNA compared to dsDNA. The sampe seems to be true for the Bioanalyzer, but such an instrument is more sensitive than our eyes looking at an agarose gel.
luc is offline   Reply With Quote
Old 10-02-2020, 12:05 PM   #5
rougaroux
Junior Member
 
Location: NYC

Join Date: Sep 2020
Posts: 3
Default

Okay, well I will give it a try with my post-ligation libraries before and after clean-up and see if there's anything to see.

Does anyone have any other suggestions of common sources of library amplification failure?
rougaroux is offline   Reply With Quote
Old 10-02-2020, 06:38 PM   #6
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 71
Default

If you're using Nanodrop on PCR products before cleanup, the "high concentration" you saw was likely just unincorporated nucleotides in the PCR mix, so that isn't useful.

If you run your cleaned up post ligation product on the Bioanalyzer you should be able to see a shift to a larger size for your ligated products compared to your original fragmented DNA. Y adapters should make this shift more noticeable since libraries ligated with Y adapter migrate slower through the gel causing them to appear larger than their actual size.

My best guess would be that there's something wrong with your custom adapters/primers or something went wrong with the prep. Maybe add a control sample that uses a non-custom adapter at ligation and p5/p7 primers for PCR.
__________________
Josh Kinman

Last edited by jdk787; 10-03-2020 at 05:43 AM.
jdk787 is offline   Reply With Quote
Reply

Tags
adapters, amplification, kapa

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 10:03 AM.


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