Protocol #07
LABORATORY OF MOLECULAR PATHOLOGY
qPCR library quantification
Using KAPA Library Quant Kit Prep:
□ 01 Thaw the KAPA SYBR FAST qPCR Master Mix. Keep it on ice.
Make sure the primers were added, or add the primers to the master mix yourself.
□ 02 Thaw Dilution Buffer and dilute it 10x with water to get a working solution.
□ 03 Thaw the six qPCR standards, keep them on ice.
□ 04 Dillute your samples 3 OOOx, 9 OOOx and 27 OOOx with the prepared lx dilution buffer. Do not use water for diluting samples. Do not pipette less than 2 \xlfor accuracy.
□ 05 Calculate how much you need and then prepare the master mix for the qPCR. Don't forget the six DNA standards and a negative control:
Master mix	lx	X
KAPA SYBR MM 2x	9,0 uL	
DEPC-H20	3,0 uL	
Diluted DNA sample	3,0 uL	-
□ 06 Pipette 12 u.L of the master mix in each of the wells in the white strips or white plates for the Roche LightCycler.
□ 07 Add 3 u.L of the diluted samples or the standards or water.
Pipette the standards from the lowest dilution to the highest (From 6 to 1).
□ 08 Seal the plate or the strips. Quick vortex and quick spin.
□ 09 Run the qPCR programme, don't forget to set the data acquisition during the annealing step:
Amplification programme	Temperature	Time
Initial denaturation	95 °C	5 min
		
Denaturation	95 °C	30 s A ft' -
Annealing + Extension	60 °C	4l)x 45 s
Melting curve
65-95 °C