SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Alternative Kapa qPCR KAPA Introductions 1 03-18-2015 06:00 AM
KAPA qPCR troubleshooting lorendarith Illumina/Solexa 2 02-12-2014 04:10 AM
KAPA qPCR kit for XL+ libraries? madseq 454 Pyrosequencing 2 08-31-2012 08:39 AM
KAPA qPCR niceday General 5 08-01-2012 12:44 AM
qPCR Kapa Kit Experimental Design sanju0891 454 Pyrosequencing 8 05-31-2012 12:33 PM

Reply
 
Thread Tools
Old 10-15-2019, 02:17 PM   #1
kgoglin
Member
 
Location: San Diego

Join Date: Dec 2014
Posts: 17
Angry qPCR standard variability with Kapa kit

We have been using Kapa's qPCR kit for Illumina library quantification for years. This year, it has given us great problems with inconsistent standards.

We have seen perfect triplicates one day and highly variable triplicates the next with the same standards. We store the standard aliquots at -20 to avoid freeze/thaws. We clean the hood prior to every run. We have tried different tubes, switching to lo bind for standard storage. We had all our pipets calibrated. We also have even changed qPCR instruments!

Has anyone else experienced this with Kapa's kit? Since we cannot find a solution, we will begin testing NEBNext's qPCR quant kit. Hopefully that will be the solution.
kgoglin is offline   Reply With Quote
Old 10-15-2019, 11:59 PM   #2
GSviral
Member
 
Location: UK

Join Date: Dec 2014
Posts: 34
Default

Hi kgoglin,

When I previously used the KAPA qPCR quantification method I also ran into the same problem from time to time. Sometimes triplicate standards could be very variable between runs even though I took all the precautions you mentioned in your post. Additionally, I used to run PhiX as a control on a KAPA run. Although I know the concentration of PhiX can be variable, sometimes I was getting absolutely wild results from a quantification run, completely out of the range I would expect the PhiX concentration to fall in. I never found out what the problem was.

Subsequently, my lab changed to NEB qPCR quantification and the last I heard it was performing really well for them so hopefully you will see some reliable results when you use it.
GSviral is offline   Reply With Quote
Old 10-16-2019, 07:41 AM   #3
kgoglin
Member
 
Location: San Diego

Join Date: Dec 2014
Posts: 17
Default

Hi GSviral,
Thank you for response. We have also begun to include PhiX as a control and it can be variable also.

We are testing the NEBNext kit today with PhiX as a control. Hoping for good results!
kgoglin 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 09:11 AM.


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