Biosearch Technologies KASP-TF V5.0 Master Mix User manual

Manual

KASP-TF V5.0 Master Mix

For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

Contents

1. Productdescriptionandspecication 3

2. Kit contents and storage 3

3. Before you start 3

4. Prepare the KASP genotyping mix 4

5. Dispensing 5

6. KASP cycling conditions 5

7. Reading KASP reactions 6
Appendix A: Performing the KASP thermal cycle on the IntelliQube instrument 7

For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

1. Productdescriptionandspecication

KASP-TF V5.0 Master Mix by LGC, Biosearch Technologies™ has been optimised for high-throughput

single nucleotide polymorphism (SNP) and insertion/deletion (indel) genotyping in Array Tape™. This 2X

mix has been designed specically for use with Array Tape on the Nexar™ in-line liquid handling and assay

processing system (Nexar system) and the IntelliQube™ instrument.

In high-throughput experiments, users typically perform thermal cycling of Array Tape using a

Hydrocycler2™ or a Soellex™. It is possible to perform thermal cycling of Array Tape on the IntelliQube

instrument; please see Appendix A for details.

KASP-TF V5.0 Master Mix is available in three pack sizes, as detailed in Table 1.

Product code Pack size Pack size

KBS-1050-201 2.5 mL KASP-TF V5.0 2X Master Mix, Standard ROX

KBS-1050-202 25 mL KASP-TF V5.0 2X Master Mix, Standard ROX

KBS-1050-204 250 mL KASP-TF V5.0 2X Master Mix, Standard ROX

KBS-1050-203

Table 1. Product codes and pack sizes for KASP-TF V5.0 Master Mix.

2. Kit contents and storage

i. KASP-TF V5.0 Master Mix – supplied at 2X concentration. Contains FAM™ and HEX™ specic

FRET cassettes, KASP Taq , dNTPs, salts, and buffer.
ii. MgCl2 – 50 mM, for optimisation of assays in particularly A/T-rich DNA regions.
iii. DMSO – for optimisation of assays in particularly G/C-rich DNA regions.

KASP-TF V5.0 Master Mix can be safely stored for 1 week at 4 °C, one year at -20 °C, or indenitely at

-80 °C. KASP-TF V5.0 Master Mix should be aliquoted for storage, and use of light-protective tubes is
recommended. Frequent freezing and thawing of KASP-TF V5.0 Master Mix is not recommended.

(250 mL)
(10 × 25 mL)

KASP-TF V5.0 2X Master Mix, Standard ROX

3. Before you start

• KASP Assay Mix should be ordered from Biosearch Technologies. Each KASP Assay Mix is specic

to the SNP or indel that is to be targeted, and consists of two competitive allele-specic primers and

one common reverse primer. Two options are available for ordering KASP Assay Mix:

– KASP-by-Design (KBD) – primers based on in silico design, no wet lab validation.
– KASP-on-Demand (KOD) – optimised and functionally validated in our genotyping laboratory.

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For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

• Thaw and vortex KASP Assay Mix and KASP-TF V5.0 Master Mix.

• Prepare DNA samples – ensure that these are at the appropriate concentration for the genome size

of your organism. Most KASP assays will function well with 5-50 ng of high quality DNA per reaction

(based on human genome size).

• If DNA is to be dried down into the Array Tape on a Nexar system with two drying heads, each drying

head should be set at 75 °C for 42000 ms on each array. These conditions will need to be optimised

if using a Nexar with greater or fewer than two DNA drying heads.

4. Prepare the KASP genotyping mix

KASP-TF V5.0 Master Mix can be dispensed at 1X or 2X concentration, depending on the format of the
DNA template, as detailed in Table 2.

DNA format Instrument KASP-TF V5.0 Master Mix

IntelliQube instrument

Wet DNA

Nexar system

Nexar system in conjunction

with the Array Tape Sample

Dry DNA

Table 2. Format for dispensing KASP-TF V5.0 Master Mix for both wet and dried down DNA samples.

Dryer (i.e. DNA samples

dried down prior to KASP

Genotyping mix dispense).

Dispense at 2X concentration
(as supplied).

Dispense at 1X concentration.
N.B. KASP-TF V5.0 2X Master
Mix must be diluted by the
addition of molecular biology

grade water, to bring the overall
nal mix concentration to 1X.

Recommended total

reaction volume

1.6 µL (See Table 3)

0.8 µL (See Table 4)

Prior to dispensing, KASP-TF V5.0 Master Mix must be combined with KASP Assay Mix according to the
ratios detailed in Tables 3 and 4 (for wet and dry DNA respectively). Exact volume requirements for each
Array Tape will be calculated by the Nexar or IntelliQube software depending on your dispensing format

(Tables 3 and 4 are for illustration only).

In Array Tape format, KASP performs well with total reaction volumes ranging from 0.8-1.6 µL.

Volume per reaction

DNA sample 0.8 µL -

KASP-TF V5.0 Master Mix 2X 0.8 µL 368.6 µL

KASP Assay Mix (72x, as supplied) 0.022 µL 10 .14 µ L

Total reaction volume 1.6 µL -

Volume for 384 reactions

(including 20% excess)

Table 3. Constituent reagent volumes for KASP genotyping reactions using wet DNA. KASP-TF V5.0 Master Mix should be used as supplied (2X).

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For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

Volume per reaction

DNA sample n/a (dry DNA) -

KASP-TF V5.0 Master Mix 1X 0.8 µL 368.6 µL

KASP Assay Mix (72x, as supplied) 0.011 µ L 5.07 µL

Total reaction volume 0.8 µL -

Table 4. Constituent reagent volumes for KASP genotyping reactions using dry DNA. KASP Array Tape Master Mix should be diluted to 1X using molecular biology

grade water.

Volume for 384 reactions

(including 20% excess)

5. Dispensing

After preparation of the KASP genotyping mix as detailed in Section 4, this should be dispensed using
the Nexar system or IntelliQube instrument. For detailed instructions on programming the dispense steps,
please refer to the appropriate instrument manual.

6. KASP cycling conditions

The KASP thermal cycling conditions detailed in Table 5 should be used when running KASP in Array Tape,
using a waterbath-based high-throughput thermal cycler (Hydrocycler2 or Soellex). Appendix A details the
thermal cycle program to use when Array Tape is to be thermally cycled on the IntelliQube instrument.

Protocol stage Temperature Duration

Stage 1 hot start Taq activation 94 °C 15 minutes × 1 cycle

94 °C 20 seconds

Stage 2 touchdown

Stage3amplication

Table 5. KASP thermal cycling conditions for reactions that will be read using the Nexar System or the IntelliQube® instrument. Please note that these conditions are
optimised specically for Array Tape and should not be used for standard PCR plates.

65 °C
(65 °C decreasing 0.8 °C per
cycle to achieve a nal annealing/
extension temperature of 57 °C)

94 °C
57 °C

60 seconds

20 seconds
60 seconds

Number of cycles

for each stage

× 10 cycles

× 26 cycles

If sufciently dened genotyping clusters are not obtained after 36 PCR cycles, Array Tape should be

further thermally cycled in increments of three cycles; the recommended recycling protocol is detailed in
Table 6. After each ‘recycling protocol’ (see Table 6), the reactions should be re-read.

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For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

Protocol stage Temperature Duration

94 °C 20 seconds

Stage1amplication

57 °C 60 seconds

Table 6. KASP further cycling conditions for for reactions that will be read using the the Nexar system or the IntelliQube instrument when additional thermal cycling

is required.

Number of cycles

for each stage

x 3 cycles

Please note, if you are using the IntelliQube to perform thermal cycling, instead of a Hydrocycler or the
Sollex, refer to the protocol detailed in Appendix A.

7. Reading KASP reactions

Reactions should be read after completion of 36 cycles (as per Table 5). KASP reactions run on the

Nexar system can be read on the Araya™ in-line uorescence detection system using standard genotyping

conditions; there is no requirement to modify any of the read settings.

To read KASP on the IntelliQube instrument, the uorophores detailed in Table 7 should be selected during

run setup.

Fluorophore IntelliQube reference

ROX Passive reference dye

FAM x-axis

HEX (or VIC*) y-axis

Table 7. Fluorophores required to read completed KASP reactions on the IntelliQube instrument.

*If the available dye sets contain VIC but not HEX, VIC can be used instead as the excitation and emission values for HEX and VIC are extremely similar.

KASP genotyping data should be plotted in a cluster plot format; typically FAM values are plotted on the
x-xis and HEX values are plotted on the y-axis. Values can be normalised using the ROX values for each

well thus removing the variable of liquid volume and leading to tighter clustering of data points. Figure 1

illustrates an example genotyping cluster plot.

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For Research Use Only. Not for use in diagnostic procedures.

Manual

Allele Y Homozygote

Heterozygote

Allele X Homozygote

NTC

KASP-TF V5.0 Master Mix

Figure 1. Example genotyping plot illustrating three clear clusters. Each data point represents the uorescence signal of an individual DNA sample. Samples of the
same genotype will have generated similar levels of uorescence and will therefore cluster together on the plot. 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).

If you have any queries, please contact Technical Support:

[email protected].

Appendix A: Performing the KASP thermal cycle on the IntelliQube instrument

If the IntelliQube is being used to perform the thermal cycle, as well as to dispense and read KASP
reactions in Array Tape, LGC, Biosearch Technologies recommend that one of the following thermal cycles
is programmed:

• KASP IntelliQube Inline protocol: 61-55 °C touchdown (Table A1, Figure A1).

• KASP IntelliQube Inline protocol: 65-57 °C touchdown (Table A2, Figure A2).

These protocols involve a hot-start activation, a 10 PCR cycle touchdown, and then 35 PCR cycles

(totalling 45 cycles, nine more than the typical KASP thermal cycle). In the third stage, a read is performed
on the IntelliQube at the end of each cycle. It is not possible to carry out a traditional KASP recycling

protocol in Array Tape on the IntelliQube, after the IntelliQube Inline thermal prole has been completed.

Instead, performing a read every cycle in Stage 3, and running more cycles as standard, enables the most
appropriate read to be selected for data analysis upon run completion.

Please note that reading the reactions every cycle does not equate to these reactions being run in real-time;
the most appropriate read is selected for end-point genotyping analysis.

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For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

Protocol stage Temperature Duration

Stage 1 hot start Taq activation 93.5 °C

Cycle 1

Cycle 2

Cycle 3

Cycle 4

Cycle 5

Stage 2 touchdown

Cycle 6

Cycle 7

Cycle 8

Cycle 9

Cycle 10

Stage3amplication

Table A1. KASP IntelliQube in-line thermal cycle protocol for 61-55 °C touchdown.

93.5 °C 20 sec

61.0 °C 1 min

93.5 °C 20 sec

60.4 °C 1 min

93.5 °C 20 sec

59.8 °C 1 min

93.5 °C 20 sec

59.2 °C 1 min

93.5 °C 20 sec

58.6 °C 1 min

93.5 °C 20 sec

58.0 °C 1 min

93.5 °C 20 sec

57.4 °C 1 min

93.5 °C 20 sec

56.8 °C 1 min

93.5 °C 20 sec

56.2 °C 1 min

93.5 °C 20 sec

55.6 °C 1 min

93.5 °C 20 sec

55.0 °C 1 min

15 min
(900 seconds)

Number of cycles

for each stage

× 1 cycle No

× 1 cycle No

x 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 35 cycles Yes

Read

Figure A1. Screenshot of the 61-55 °C protocol when programmed in the IntelliQube software.

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For Research Use Only. Not for use in diagnostic procedures.

Manual

KASP-TF V5.0 Master Mix

Protocol stage Temperature Duration

Stage 1 hot start Taq activation 93.5 °C

Cycle 1

Cycle 2

Cycle 3

Cycle 4

Cycle 5

Stage 2 touchdown

Cycle 6

Cycle 7

Cycle 8

Cycle 9

Cycle 10

Stage3amplication

Table A2. KASP IntelliQube in-line thermal cycle protocol for 65-57 °C touchdown.

93.5 °C 20 sec

65.0 °C 1 min

93.5 °C 20 sec

64.2 °C 1 min

93.5 °C 20 sec

63.4 °C 1 min

93.5 °C 20 sec

62.6 °C 1 min

93.5 °C 20 sec

61.8 °C 1 min

93.5 °C 20 sec

61.0 °C 1 min

93.5 °C 20 sec

60.2 °C 1 min

93.5 °C 20 sec

59.4 °C 1 min

93.5 °C 20 sec

58.6 °C 1 min

93.5 °C 20 sec

57.8 °C 1 min

93.5 °C 20 sec

57.0 °C 1 min

15 min
(900 seconds)

Number of cycles

for each stage

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 1 cycle No

× 35 cycles Yes

Read

Figure A2. Screenshot of the 61-55 °C protocol when programmed in the IntelliQube software.

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For Research Use Only. Not for use in diagnostic procedures.

For Research Use Only. Not for use in diagnostic procedures

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