qPCR Kapa Kit Experimental Design

[sanju0891]

Hello folks,

In our lab we are thinking to use the Kappa library quant kit for Roche GS FLX System on a Lightcycler 480 for the first time.

in the protocol of the manufacturer (Kappa) they suggest to do a triplicate for every sample and standard. so is it really necessary to do triplicates for all samples we have. we have like 40 samples to be sequenced, so if i do a triplicate for each sample one 96 well plate wont be sufficient.

did somebody also try the 10µl reaction voulme instead of 20µl? is there any difference between them.

and is it necessary to do 3-4 serial dilutions of the samples. which dilutions have worked better (1:500 or 1:1000)?

pls help me with these...........

[ETHANol]

No it is overkill, but it also depends on how well you can pipet and the quality of your pipets. I would choose one dilution and you could do duplicates to be safe. Use the least diluted sample that falls within the standard curve.

[sanju0891]

so i do not need to do triplicates for every sample?

sorry for asking this but im really confused about this. what protocol should i follow correctly

[sanju0891]

should i have to prepare the 6 DNA standards triplicates for each plate i run or only one of the six is enough for each plate????

so many questions regarding this? Please can anyone tel mein detail how to do this procedure for my 40 samples

[Tom Barker]

you will need to prepare all six standards as they are used for the standard curve which is used to calculate the concentration of your samples. You can set these up in triplicate but i only set duplicates. I've never used the light cycler before but on the Step One instrument i use you can choose to exclude wells from the standard curve if for example you have made a pipetting error when setting up the plate. If your careful with your pipetting you shouldn't need to set up triplicates.

[RCJK]

For rapid libraries I do 1:500 and 1:1000 in triplicate and leave out the first standard as it's too high. If your samples are amplicons you'll need to start at a higher dilution.

[mohdsos]

Guys would you dilute the 10^7 tube, or the original tube, because I am trying to pool 6 libraries.

[sanju0891]

we dilute the original to further.

[genomeseeker]

We've found these dilutions work over 90% of the time and we do 10uL reactions in triplicate
rapid libs = 1:400
PE libs = 1:10,000
Amplicon libs = 1:50,000