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kgoglin 10-15-2019 01:17 PM

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.

GSviral 10-15-2019 10:59 PM

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.

kgoglin 10-16-2019 06:41 AM

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!

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