KAPABIOSYSTEM library quantification kits

[elena.85]

High everybody!
I'm a new entry of the forum and I would like to know if someone have already tried the library quantification kit above!
What's your opinion about? Do you find it useful for pooling samples and multiplexing? Do you replace the emPCR titration with this quantification?

I have already post this question in the Illumina/Solexa section because I will make experiments with both the technologies...

Thank you for your help!

Elena

[TonyBrooks]

High everybody!
I'm a new entry of the forum and I would like to know if someone have already tried the library quantification kit above!
What's your opinion about? Do you find it useful for pooling samples and multiplexing? Do you replace the emPCR titration with this quantification?

I have already post this question in the Illumina/Solexa section because I will make experiments with both the technologies...

Thank you for your help!

Elena

Yes. I use the Kapa kit. It's pretty good, and more importantly quicker and cheaper than titration.

[elena.85]

Yes. I use the Kapa kit. It's pretty good, and more importantly quicker and cheaper than titration.

Thank you!
So you replace the titration with this qPCR quantification, right?
Did you start to use the kapa kit since your first experiment or did you first use the titration to set the initial conditions and then you trusted the qPCR?

Thank you again!

Elena

[TonyBrooks]

Thank you!
So you replace the titration with this qPCR quantification, right?
Did you start to use the kapa kit since your first experiment or did you first use the titration to set the initial conditions and then you trusted the qPCR?

Thank you again!

Elena

We use a control sample on every run. It's a sample that has been emPCR'd a few times and consistently gives us 10% enrichment at 0.5cpb. At 1 million moleucles/µL, it lies in the middle of our standard curve (using the standards provided by Kapa). After the qPCR, we then dilute our test samples to the same as the control sample and use this for emPCR.
There is some variablity, but usually only +/- 3% in terms of enrichment.
Also, we've noticed that different library types can vary in emPCR. Our amplicon libraries need 2X the amount of cpb when compared to rapid DNA libraries.

[pmiguel]

Also, we've noticed that different library types can vary in emPCR. Our amplicon libraries need 2X the amount of cpb when compared to rapid DNA libraries.

Would that not just be because you denature the rapid DNA libraries and thus each strand serves as separate amplicon, whereas there is no requirement to do this for amplicons?

--
Phillip

[TonyBrooks]

Would that not just be because you denature the rapid DNA libraries and thus each strand serves as separate amplicon, whereas there is no requirement to do this for amplicons?

--
Phillip

This is my reasoning also.
However, we also denatured our amplicons once and didn't notice any difference. Surely in this case, it should perform like the rapid library - which it didn't. We'd still get amplification from the - strand as the amplification primer would anneal to and generate the + strand during the 1st cycle of PCR.

[pmiguel]

Strange.
Any other hints (lib QC?) for getting the qPCR to give you reliable emPCR enrichments?

We always use it, but find it very hit-or-miss and end up doing faux-titrations (do one MV emPCR, adjust concentration for more the next day if necessary).

Actually we recently determined that the strip caps we had been using (inexplicably) to seal our 96-well plates were apparently not sealing well enough and allowing evaporation/emulsion breakage. Since the new emulsion oils were introduced, breakage looks nothing like what is portrayed in the manual -- or at least the breakage has modes where it does not. Basically any clear material on the bottom is indicative of at least partial emulsion breakage. We had some wells with a bubble of oil on the bottom less than 1 mm across. Nevertheless, even a little breakage can cause issues that drastically impact enrichment percentages.

--
Phillip

[MissDNA]

Has anyone ever counted the amount of beads/tube that come in the emPCR kits?
I was talking to someone that was having a lot of enrichment issues in the lab (over 20%), and she told me she started counting the beads/tube. She got tubes with 28 million beads instead of 35 million. Obviously for those who make their cpb calculations based on 35 million beads, having only 28 makes a big difference.
In our lab we do traditional titration but using only 2 points and a lot of times we feel like is a guessing game.

[pmiguel]

We usually end up with more beads than expected after breaking. (We always collect an aliquot of pre-enriched beads to count.)

I was wondering where those extra beads came from. Looks like they are getting stolen from kits heading to Brazil. Sorry about that!

--
Phillip

[MissDNA]

Hahaha I did laugh.

Now that you mentioned, you had some titrations where we recovered more beads than the supposed input.

Could you explain more about the faux-titrations you do?

[pmiguel]

Well, I am not sure I recommend them. Not my idea. But a tech in the lab was not able to get the SV emPCRs to match his MV emPCR enrichment numbers. So he would do qPCR, and try a couple of MV emPCR reactions based on his qPCR results. If all went perfectly, he would have enough beads for his run. If not, he would adjust his amount of starting library according to the enrichments he got with the first set of emPCRs and do another set of emPCRs.

I think because we were having an undiagnosed emPCR breaking issues caused by the strip caps being used, day to day results would also vary dramatically.

--
Phillip

[MissDNA]

We were never able to match SV to MV numbers. Is even more unpredictable than SV to LV. We use MV only for 4 regions and SV for 8 regions. We also don't use qPCR. As I mentioned before we do a traditional titration but using only 2 SV emulsions. Your lab's tech way seems more complicated than ours but if is working, great!