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User guide
KASP end-point genotyping trial kit
For Research Use Only. Not for use in diagnostic procedures.
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User guide
KASP end-point genotyping trial kit
Contents
1. Introduction
2. Kit contents
3. Experimental procedure for performing KASP end point genotyping reactions
3.1 KASP genotyping reactions
3.2 Kit assembly
3.3 KASP genotyping mix assembly
3.4 Experimental procedure
3.5 Thermal cycling conditions
3.6 Additional cycling conditions
4. Ordering information
5. Technical support
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User guide
KASP end-point genotyping trial kit
1. Introduction
The KASP™ genotyping trial kit, from LGC, Biosearch Technologies™, is supplied as an introduction
to the use of KASP chemistry. It enables the user to trial the chemistry in their own laboratory using
existing equipment.
In general, any qPCR instrument or plate reader that is capable of reading FRET at the wavelengths in
Table 1 should be compatible with KASP.
KASP has been validated on a wide range of qPCR instruments including:
• ABI 7500
• ABI 7300
• ABI 7900
• ABI ViiA7
• Roche LC480
• Agilent Mx3000P/3005P
• illumina EcoRT
• BIO-RAD CFX
Please visit our website to view a full list of the instruments that have been validated for KASP
chemistry to date, to access our instrument-specic manuals, and to determine the correct version of
KASP-TF Master Mix for your machine.
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User guide
KASP end-point genotyping trial kit
2. Kit contents
This kit contains a KASP genotyping assay comprising of KASP Assay Mix, KASP-TF Master Mix and
DNA:
1. 1 × 96-well microtitre plate containing 33 DNA samples (and 3 no-template controls) pre-diluted to
concentration range appropriate for KASP genotyping reactions.
2. 1 × 500 µL tube of KASP-TF Master Mix at 2x concentration (sufcient for 100 reactions
at 10 µL).
3. 1 × tube of KASP Assay Mix (at 72x concentration).
Customer requirements
1. FRET-capable plate reader (see Table 1 for details of the excitation and emission
values of the uorophores)
2. One empty PCR plate of the type intended for use in the customer’s genotyping process
3. PCR grade water
4. Optically clear plate seal
Fluorophore Excitation (nm) Emission (nm)
FAM 485 520
HEX 535 556
ROX 575 610
Table 1. Fluorophores used in the KASP chemistry and their respective excitation and emission wavelengths.
3. Experimental procedure for performing KASP end-point genotyping reactions
3.1 KASP genotyping reactions
The following reagents are used to prepare the KASP reaction:
• 2x KASP-TF Master Mix
• 72x KASP Assay Mix
• DNA samples (sample DNA is provided at the correct concentration for using at a 2x dilution)
• Water to be added if necessary to make the nal reaction volume.
3.2 Kit assembly
A 5 µL total reaction volume is required for 384-well plates and a 10 µL total reaction volume for
96-well plates.
Table 2 is an example of how to construct 60 reactions for KASP for both 384- and 96-well plate
formats. This will enable you to make enough genotyping mix for the 33 samples and 3 no template
controls (NTCs), with an additional spare volume.
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KASP end-point genotyping trial kit
3.3 KASP genotyping mix assembly
Component Wet DNA method (µL) for 60 reactions
Plate format 384-well plate 96-well plate
2x KASP-TF Master Mix 150 300
Assay Mix 4.2 8.4
Water N/A N/A
Total reaction volume 5 10
Total 300 600
Table 2. An example of KASP reaction assembly
3.4 Experimental procedure
1. Thaw the KASP-TF Master Mix and KASP Assay Mix tubes. Vortex each tube, and store on ice.
2. Thaw the DNA plate. Briey vortex and spin down the plate.
3. Use a single- or multi-channel pipette to transfer DNA to the empty reaction plate (supplied by
customer).
For a 96-well plate: transfer 5 µL of the DNA provided to each well.
For a 384-well plate: transfer 2.5 µL of the DNA provided to each well.
4. Prepare the genotyping mix (2x KASP-TF Master Mix plus Assay Mix) as outlined in Table 2. Add
the required amount of genotyping mix to each DNA sample in the reaction plate. A single- or multichannel pipette can be used for this procedure.
For a 96-well plate: add 5 µL of the prepared genotyping mix to each DNA sample.
For a 384-well plate: add 2.5 µL of the prepared genotyping mix to each DNA sample.
5. Seal the plate with an optically clear seal.
6. Centrifuge the plate at a minimum of 550 x g. Do not spin the plates at a higher speed than is
recommended for your rotor.
3.5 Thermal cycling conditions
The KASP chemistry can be used with any standard thermal cycler. The thermal cycling conditions are
detailed in Table 3. Please note that a two-step touchdown PCR method is used, with the elongation
and annealing steps incorporated into a single step. It is recommended to cycle the reaction plates
as soon as possible after the DNA and genotyping mix have been dispensed. If this is not feasible,
prepared plates can be stored for up to 1 hour at 4 °C. Plates should be sealed with an optically clear
seal immediately after dispensing to prevant any evaporation.
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KASP end-point genotyping trial kit
Step Description Temperature Time Number of cycles per step
1 Activation 94 ºC 15 min 1
Denaturation 94 ºC 20 sec
2
Annealing/elongation 61-55 ºC
Denaturation 94 ºC 20 sec
3
Annealing/elongation 55 ºC 60 sec
Table 3. Thermal cycling conditions for the KASP chemistry
60 sec
(drop 0.6 ºC per cycle)
After thermal cycling, read the plate.
Please note: All plates should be read below 40 ºC. If the plate is not read below 40 ºC , it will not be
possible to analyse the genotyping data.
10 cycles
26 cycles
3.6 Additional cycling conditions
If you have not obtained clear genotyping clusters, the plate should be thermally cycled for an
additional 3 cycles and read again. Please see Table 4 for recycling conditions.
Step Temperature Time Number of cycles per step
Denaturation 94 ºC 20 sec
Annealing/
elongation
Table 4. Conditions for further thermal cycling of the KASP chemistry
57 ºC 60 sec
3 cycles
Further cycling and reading can be performed until tight genotyping clusters have been attained.
Figure 1. Example genotyping plot illustrating three clear clusters. The FAM allele is reported on the X axis (blue data points)
and the HEX allele is reported on the Y axis (red data points). The green data points represent heterozygous samples and
the black data points are the No Template Controls (NTCs). The 96-well plate layout indicates expected genotyping results
for KASP genotyping trial kit DNA samples.
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KASP end-point genotyping trial kit
Once the thermal cycling have been performed, we highly recommend storing the plate in a dark fridge
(~4 °C for a maximum of 1 week) until the data has been analysed. This will allow you to then perform
additional read(s) or recycle steps if required, to ensure that you have obtained the best possible data.
Note: This protocol pertains to all three of the trial kit part numbers. The only difference between the
three kits is the concentration of the passive reference dye ROX in the KASP-TF Master Mix, which
varies based upon the type of reader being used. To determine which trial kit is right for you, please
contact our technical support team [email protected] or visit our website.
4. Ordering information
Product code Description
KBS-1050-164 KASP-TF V4.0 Trial Kit (Standard ROX)
KBS-1050-165 KASP-TF V4.0 Trial Kit (High ROX)
KBS-1050-166 KASP-TF V4.0 Trial Kit (Low ROX)
5. Technical support
For any queries about running KASP in your laboratory, please contact [email protected].
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