KAPA SYBR® FAST qPCR¶

Protocol¶

Any existing qPCR assay performed efficiently using standard cycling conditions may be converted to a fast qPCR assay with KAPA SYBR FAST qPCR Kits. Typically, re-optimization of reaction parameters is required.

qPCR is performed at the MIT BioMicro Center on a Roche LightCycler 480 using SYBR Green MasterMix. Roche’s documentation is here.

1. Master Mix Preparation¶

Ensure all reaction components are properly thawed and mixed.
Prepare a master mix containing the appropriate colume of all reaction components common to all or a subset of reactions to be performed.
Always include a No Template Control (NTC) to allow for detection of contamination of reaction components.
Calculate the required volume of each component based on the following tables:

Component	ROX	No ROX	Final conc.
PCR-grade water	Up to 20 μL	Up to 20 μL	N/A
KAPA SYBR FAST qPCR Master Mix (2X) Universal	10 µL	10 µL	1X
10 µM forward primer	0.4 µL	0.4 µL	200 nM
10 µM reverse primer	0.4 µL	0.4 µL	200 nM
Template DNA	As required	As required	<20 ng
50X ROX High/Low (as required)	0.4 µL	--	1X

2. Reaction Setup¶

Transfer the appropriate volumes of qPCR master mix, template, and primers to each well of a PCR plate/tube(s).
Cap or seal the reaction plate/tube(s) and centrifuge briefly.

3. qPCR¶

If applicable, select fast mode on the instrument.
Confirm that the qPCR protocol to be used conforms to the following parameters:

Detection Format	Block Type	Reaction Volume
SYBR Green	96 well	10 - 25 µL
SYBR Green	384 well	3 - 20 µL
Program Name	Cycles	Analysis Mode
Pre-incubation	1	None
Amplification	40	Quantification
Melting Curve	1	Melting Curves
Cooling	1	None
Program Name	Target (°C)	Aquisition Mode	Hold (hh:mm:ss)
Pre-incubation	95	None	00:03:00
Amplification	95	None	00:00:10
	Primer dependent	None	00:00:20
	72	Single	00:00:01
Melting curve	95	None	00:00:05
	65	None	00:01:00
	97	Continuous	5-10 acq/ °C
Cooling	40	None	00:00:10

4. Data Analysis¶

Data analysis is dependent on experimental design. Refer to the instrument guidelines for more information on how to perform the appropriate data analysis.