KAPA Library Preparation kit with Real

KAPA Library Preparation kit with Real
Technical Data Sheet
KAPA Library Preparation Kits
Kit codes and components
with Real-Time PCR Library Amplification
KAPA Library Preparation Kit with Real-Time
PCR Library Amplification - Illumina
Illumina series
Product Description
KK8220
10 reactions
The KAPA Library Preparation Kit provides all of the enzymes and
reaction buffers required for constructing libraries from fragmented
dsDNA via the following steps:
- End Repair Enzyme Mix (50 µL)
- 10X End Repair Buffer with dNTPs (100 µL)
- A-Tailing Enzyme (30 µL)
- 10X A-Tailing Buffer (50 µL)
- DNA Ligase (50 µL)
- 5X Ligation Buffer (100 µL)
- 2X KAPA HiFi HotStart Real-Time PCR
Master Mix (250 µL)
- 4 x Fluorescent Standards (1500 µL each)
1. End repair: Produce blunt-ended, 5’-phosphorylated fragments.
KAPA Library Preparation Kit with Real-Time
PCR Library Amplification - Illumina
2. A-tailing: Add dAMP to the 3’-ends of the dsDNA library fragments.
3. Adaptor ligation: Ligate dsDNA adaptors with 3’-dTMP overhangs to
library fragments.
4. Library amplification: Real-time amplification of library
fragments carrying appropriate adaptor sequences on both ends.
Reaction buffers are supplied in convenient, concentrated “master mix”
formats comprising all of the required reaction components except
oligonucleotide adaptors and PCR primers. Similarly, a single enzyme
mixture is provided for each step of the library construction process,
reducing the number of pipetting steps.
High fidelity PCR is used to selectively enrich library fragments carrying
appropriate adaptor sequences and to amplify the amount of DNA
prior to sequencing. During PCR enrichment of libraries, minimizing
amplification bias is critical to ensure more uniform sequence coverage.
Amplification bias occurs when a DNA polymerase is unable to amplify
all targets within a complex population of library DNA with equal
efficiency. Bias is further exacerbated when libraries are over-amplified.
KAPA HiFi Real-Time PCR Library Amplification Kits are designed to
address both sources of PCR-induced bias. The novel KAPA HiFi DNA
Polymerase, engineered for high fidelity and processivity, is capable of
balanced amplification of complex library DNA. Real-time monitoring
of library amplification provides additional information required
to optimize the number of amplification cycles and minimize overamplification. The benefits of performing high fidelity, real-time PCR for
next-generation sequencing library amplification include:
•
Real-time monitoring of amplification allows precise control over
the optimal number of PCR cycles.
•
Real-time amplification workflows are amenable to automation.
•
Real-time amplification plots provide quality metrics for
individual enriched libraries, eliminating expensive and timeconsuming post-enrichment gel electrophoresis and identifying
inconsistencies in library preparation.
•
KK8221
50 reactions
- End Repair Enzyme Mix (250 µL)
- 10X End Repair Buffer with dNTPs (500 µL)
- A-Tailing Enzyme (150 µL)
- 10X A-Tailing Buffer (250 µL)
- DNA Ligase (250 µL)
- 5X Ligation Buffer (500 µL)
- 2X KAPA HiFi HotStart Real-Time PCR
Master Mix (1250 µL)
- 4 x Fluorescent Standards (1500 µL each)
Storage and handling
Store all components at -20 °C. KAPA HiFi HotStart Real-Time PCR
Master Mix (2X) and fluorescent standards 1 – 4 are light sensitive
and should be protected from light during storage, thawing, and
reaction setup. Please refer to Section 6 for full details.
Quick Notes
 Reaction components should be mixed fresh and used on
the same day.
Reaction
cleanup between successive enzymatic reactions
may be accomplished via your method of choice. We
recommend Agencourt Ampure XP Beads or Qiagen
MinElute Reaction Cleanup Kits.
Optimal
real-time library amplification corresponds to
the region between fluorescent standard 1 and 3. The
termination cycle number should be adjusted accordingly
without the requirement for performing gel electrophoresis
(see Figure 1, page 9).
 It is critical that the correct data acquisition temperature is
adhered to, to minimize background fluorescence due to
inter- and intra-primer interaction (see Table 1, page 12).
When
using primers that differ in sequence from those
listed in Table 1, we recommend performing gradient PCR to
optimize the annealing temperature.
Seamless integration with KAPA Library Quantification Kits.
KAPA HiFi Real-Time PCR Library Amplification Kits contain KAPA HiFi HotStart Real-Time PCR Master Mix (2X), a ready-to-use cocktail containing
all components for PCR, except primers and template. The 2X Master Mix contains KAPA HiFi HotStart DNA Polymerase in a proprietary reaction
buffer, dNTPs, MgCl2 (2.5 mM at 1X), SYBR® Green I dye and stabilizers. Four fluorescent standards are supplied, and are used to define a window
for optimal amplification (Figures 1 and 2).
This kit is primarily intended for the construction of genomic, paired-end, and paired-end multiplex (indexed/barcoded) Illumina DNA libraries,
but may be used for other applications requiring efficient end-repair, A-tailing, ligation, and/or library amplification steps.
www.kapabiosystems.com
Version 3.11
KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
1. End Repair
Materials required but not supplied in this kit:
Reaction tubes. Reactions may be assembled and processed in PCR plates, PCR tubes, or microcentrifuge tubes.
Pipette tips. We strongly recommend the use of high quality filter-plugged tips to prevent contamination of reagents and library samples.
Reaction cleanup. We recommend Agencourt AMPure XP Beads (cat. # A63881) or Qiagen MinElute Reaction Cleanup Kits (cat. # 28204)
for this purpose. AMPure XP Beads require the preparation of an elution buffer (10 mM TRIS-Acetate, pH 8.0, reagent grade water).
1.1
Assemble the End Repair reaction:
1.2
1.3
Water to 100 µL
10X End Repair Buffer End Repair Enzyme Mix 1 - 5 µg sheared dsDNA Total
x µL
10 µL
5 µL
1 - 85 µL
100 µL
Incubate for 30 min @ 20 °C
Proceed immediately to cleanup.
End Repair Cleanup
We recommend either Agencourt AMPure XP Beads, or Qiagen MinElute Reaction Cleanup Kits. Below are suggested protocols for the
recommended methods:
AMPure XP Beads
1.4
1.5
Ensure that the AMPure XP Beads are equilibrated to room temperature, and that they are thoroughly resuspended.
Add AMPure Beads to the End Repair reaction:
End Repair reaction
AMPure XP Beads
Total
1.6
1.7
1.8
1.9
1.10
1.11
1.12.
1.13
1.14
1.15
100 µL
160 µL
260 µL
Mix thoroughly on a vortex mixer or by pipetting up and down at least ten times.
Incubate at room temperature for 15 minutes to allow DNA to bind to the beads.
Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
Carefully remove and discard 255 µL of the liquid. Take care not to disturb or discard any of the beads. Some liquid may remain visible
in the tube/well.
Keeping the tube/plate on the magnetic stand and without disturbing the beads, wash the beads in 200 µL of 80% EtOH for at
least 30 seconds.
Carefully remove and discard the ethanol without disturbing the beads, and repeat the process for a total of 2 washes in 80% EtOH.
Remove the tube/plate from the magnetic stand, and allow the beads to dry at room temperature for 15 minutes.
Resuspend the beads thoroughly in 32.5 µL elution buffer, and incubate at room temperature for 2 minutes to release the DNA from
the beads.
Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
Recover the DNA in 30 µL of supernatant and transfer to the tube/well in which you intend to perform the A-tailing reaction.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
1. End Repair (cont.)
OR
Qiagen MinElute Reaction Cleanup Kit
The procedure for reaction cleanup using Qiagen MinElute columns described below is abbreviated and is intended for users who are already
familiar with the method. Please consult the documentation provided by the supplier for full details.
1.4 If the End Repair reaction was performed in a PCR tube/plate, transfer the End Repair reaction to a microcentrifuge tube before proceeding.
1.5 Follow the MinElute protocol:
End Repair reaction
Add Buffer ERC
Total
1.6
1.7
1.8
1.9
100 µL
300 µL
400 µL
Apply the mixture to a column and centrifuge or apply vacuum as appropriate. Discard the flow-through.
Wash with 750 µL Buffer PE. Discard the flow-through.
Centrifuge for 2 minute at ≥ 10,000 x g to remove all traces of ethanol.
To elute, transfer the column to a clean, sterile microcentrifuge tube and add 31 µL buffer EB. Incubate for 1 minute at room
temperature, and centrifuge to recover ~30 µL.
**Safe Stopping Point**
If you are not proceeding to A-Tailing immediately, the protocol can be safely stopped here. Store at -20 ºC for up to seven days.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
2. A-Tailing
2.1 Assemble the A-Tailing reaction:
Water
10X A-Tailing Buffer A-Tailing Enzyme
End repaired DNA
Total
2.2
2.3
12 µL
5 µL
3 µL
30 µL
50 µL
Incubate for 30 min @ 30 °C
Proceed immediately to cleanup.
A-Tailing Cleanup
We recommend either Agencourt AMPure XP Beads, or Qiagen MinElute Reaction Cleanup Kits. Below are suggested protocols for the
recommended methods:
AMPure XP Beads
2.4
2.5
Ensure that the AMPure XP Beads are equilibrated to room temperature, and that they are thoroughly resuspended.
Add AMPure XP Beads to the End Repair reaction:
A-Tailing reaction
Add AMPure XP Beads
Total
2.6
2.7
2.8
50 µL
90 µL
140 µL
Mix thoroughly on a vortex mixer or by pipetting up and down at least ten times.
Incubate at room temperature for 15 minutes to allow DNA to bind to the beads.
Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
2.9 Carefully remove and discard 135 µL of the liquid. Take care not to disturb or discard any of the beads. Some liquid may remain visible
in the tube/well.
2.10 Keeping the tube/plate on the magnetic stand and without disturbing the beads, wash the beads in 200 µ L of 80% EtOH for at least
30 seconds.
2.11 Carefully remove and discard the ethanol without disturbing the beads, and repeat the process for a total of 2 washes in 80% EtOH.
2.12 Remove the tube/plate from the magnetic stand, and allow the beads to dry at room temperature for 15 minutes.
2.13 Resuspend the beads thoroughly in 32.5 µL elution buffer, and incubate at room temperature for 2 minutes to release the DNA from
the beads.
2.14 Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
2.15 Recover the DNA in 30 µL of supernatant and transfer to the tube/well in which you intend to perform the adaptor ligation reaction.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
2. A-Tailing (cont.)
OR
Qiagen MinElute Reaction Cleanup Kit
The procedure for reaction cleanup using Qiagen MinElute columns described below is abbreviated and is intended for users who are
already familiar with the method. Please consult the documentation provided by the supplier for full details.
2.4
If the end repair reaction was performed in a PCR tube/plate, transfer the End Repair reaction to a microcentrifuge tube before
proceeding.
2.5 Follow the MinElute protocol:
A-Tailing Reaction
Add Buffer ERC
Total
2.6
2.7
2.8
2.9
50 µL
300 µL
350 µL
Apply the mixture to a column and centrifuge or apply vacuum as appropriate. Discard the flow-through.
Wash with 750 µL Buffer PE. Discard the flow-through.
Centrifuge for 2 minute at ≥ 10,000 x g to remove all traces of ethanol.
To elute, transfer the column to a clean, sterile microcentrifuge tube and add 31 µL buffer EB. Incubate for 1 minute at room
temperature, and centrifuge to recover ~30 µL.
**Safe Stopping Point**
If you are not proceeding to Adaptor Ligation immediately, the protocol can be safely stopped here. Store at -20 ºC for up to seven days.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
3. Adaptor Ligation
3.1 Assemble the Adaptor Ligation reaction:
5X Ligation Buffer
DNA Ligase
DNA Adaptor (30 µM)*
A-Tailed DNA
Total
10 µL
5 µL
5 µL
30 µL
50 µL
*Not supplied
3.2 Incubate for 15 min @ 20 °C
3.3 Proceed immediately to cleanup.
Adaptor Ligation Cleanup
Note:
If using AMPure XP Beads for DNA recovery and purification, we recommend that you follow the protocol outlined below to perform
a total of TWO successive cleanup procedures after adaptor ligation. If you are using the MinElute Reaction Cleanup Kit, then a single cleanup procedure is recommended.
AMPure XP Beads
First AMPure XP Bead Cleanup
3.4 Ensure that the AMPure XP Beads are equilibrated to room temperature, and that they are thoroughly resuspended.
3.5 Add AMPure Beads to the End Repair reaction:
Adaptor Ligation reaction
50 µL
Add AMPure XP Beads
50 µL
Total
100 µL
3.6 Mix thoroughly on a vortex mixer or by pipetting up and down at least ten times.
3.7 Incubate at room temperature for 15 minutes to allow DNA to bind to the beads.
3.8 Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
3.9 Carefully remove and discard 95 µL of the liquid. Take care not to disturb or discard any of the beads. Some liquid may remain visible in
the tube/well.
3.10 Keeping the tube/plate on the magnetic stand and without disturbing the beads, wash the beads in 200 µL of 80% EtOH for at least
30 seconds.
3.11 Carefully remove and discard the ethanol without disturbing the beads, and repeat the process for a total of 2 washes in 80% EtOH.
3.12 Remove the tube/plate from the magnetic stand, and allow the beads to dry at room temperature for 15 minutes.
3.13 Resuspend the beads thoroughly in 52.5 µL elution buffer, and incubate at room temperature for 2 minutes to release the DNA from
the beads.
3.14 Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
3.15 Recover the DNA in 50 µL of supernatant and transfer to the tube/well in which you intend to perform the second cleanup procedure
(see below).
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
3. Adaptor Ligation (cont.)
Second AMPure XP Bead Cleanup
3.16 Ensure that the AMPure XP Beads are equilibrated to room temperature, and that they are thoroughly resuspended.
3.17 Add AMPure Beads to the End Repair reaction:
Library DNA from first cleanup 50 µL
50 µL
Add AMPure XP Beads
100 µL
Total
3.18 Mix thoroughly on a vortex mixer or by pipetting up and down at least ten times.
3.19 Incubate at room temperature for 15 minutes to allow DNA to bind to the beads.
3.20 Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
3.21 Carefully remove and discard 95 µL of the liquid. Take care not to disturb or discard any of the beads. Some liquid may remain visible in
the tube/well.
3.22 Keeping the tube/plate on the magnetic stand and without disturbing the beads, wash the beads in 200 µL of 80% EtOH for at least
30 seconds.
3.23 Carefully remove and discard the ethanol without disturbing the beads, and repeat the process for a total of 2 washes in 80% EtOH.
3.24 Remove the tube/plate from the magnetic stand, and allow the beads to dry at room temperature for 15 minutes.
3.25 Resuspend the beads thoroughly in 32.5 µL elution buffer, and incubate at room temperature for 2 minutes to release the DNA from
the beads.
3.26 Capture the beads by placing the tube/PCR plate on an appropriate magnetic stand at room temperature for 15 minutes or until the
liquid is completely clear.
3.27 Recover the DNA in 30 µL of supernatant and transfer to the tube/well in which you intend to store the library DNA until you carry out
size selection.
OR
Qiagen MinElute Reaction Cleanup Kit
The procedure for reaction cleanup using Qiagen MinElute columns described below is abbreviated and is intended for users who are
already familiar with the method. Please consult the documentation provided by the supplier for full details.
3.4 If the end repair reaction was performed in a PCR tube/plate, transfer the End Repair reaction to a microcentrifuge tube before proceeding.
3.5 Follow the MinElute protocol:
Adaptor Ligation reaction
Add Buffer ERC
Total
3.6
3.7
3.8
3.9
50 µL
300 µL
350 µL
Apply the mixture to a column and centrifuge or apply vacuum as appropriate. Discard the flow-through.
Wash with 750 µL Buffer PE. Discard the flow-through.
Centrifuge for 2 minute at ≥ 10,000 x g to remove all traces of ethanol.
To elute, transfer the column to a clean, sterile microcentrifuge tube and add 31 µL buffer EB. Incubate for 1 minute at room temperature,
and centrifuge to recover ~30 µL.
**Safe Stopping Point**
If you are not proceeding to Size Selection immediately, the protocol can be safely stopped here. Store at -20 ºC for up to seven days.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
4. Size Selection
It is important to remove unligated adaptor molecules prior to library amplification to prevent the formation of “adaptor dimers” and
other short adaptor-derived molecules, which may cause problems downstream during cluster amplification and sequencing. For many
sequencing libraries/protocols, it is also helpful to select a relatively narrow and precisely defined size range of library fragments. Depending on
your needs and options, you may choose to perform this size selection via a variety of common methods including:
“SPRI-bead” size selection.
“Double-SPRI-bead” size selection.
Manual agarose gel electrophoresis, excision, and purification.
Automated DNA size selection and collection (e.g. Sage Science Pippin Prep™).
Due to the wide variety of viable alternatives for this procedure we do not provide any specific protocols for size selection.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
5. Real-Time Library Amplification
5.1 Overview of real-time high fidelity amplification of next-generation DNA sequencing libraries.
Figure 1. Libraries are amplified using a SYBR® Green-based real-time, high fidelity PCR master mix (left panel). Four triplicate
wells of the PCR plate contain fluorescent reference standards representing a range of distinct DNA concentrations. Reactions
terminated between standards 1 and 3 represent the optimal library amplification range (grey box), depicted here from cycles
10 - 14. A gel image of a typical library stopped at different amplification cycles is shown in the right panel. Low- and highmolecular weight artifacts increase progressively with additional cycles.
0
45 000
5
10
15
Time (min)
20
25
30
35
40
Fluorescent Standard 4
40 000
Fluorescence
35 000
Fluorescent Standard 3
30 000
25 000
Fluorescent Standard 2
20 000
15 000
Fluorescent Standard 1
10 000
5 000
0
0 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Cycle
Figure 2a. Superimposed amplification plots for reactions
terminated at the lower bound (hashed line, cycle 10) or upper
bound (solid line, cycle 14) of the targeted concentration range
(grey box). Library amplification reactions should ideally be
terminated anywhere within the indicated target concentration
range.
9
Figure 2b. Example of real-time high fidelity amplification of
multiple libraries. 20 libraries, spanning a ~64-fold concentration
range (6 cycles), were simultaneously amplified and terminated
after 14 cycles. 14 of the 20 libraries fall within the targeted
amplification range. The remaining 6 libraries could either
be used as is, noting that they may be outside the optimal
concentration range, or they could be re-amplified individually
or in high- or low-concentration groups.
KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
5. Real-Time Library Amplification (cont.)
5.2 Preparation
Note: KAPA HiFi HotStart Real-Time PCR Master Mix (2X) and fluorescent standards 1 - 4 are light sensitive and should be protected from
light.
Thaw the primers required for PCR enrichment (PCR primers are not supplied in the KAPA Library Preparation Kit), a tube of KAPA HiFi
HotStart Real-Time PCR Master Mix (2X), and fluorescent standards 1 - 4 at room temperature.
Mix and briefly centrifuge the thawed KAPA HiFi HotStart Real-Time PCR Master Mix (2X), primer and fluorescent standards 1 - 4.
Thaw, gently mix, and briefly centrifuge the adaptor-ligated, size-separated purified library DNA for 5 seconds at 600 xg.
Pre-program the real-time thermal cycler with the appropriate cycling protocol (see Table 1) for each specific set of library
amplification PCR primers.
5.3 Reaction setup
Each plate must contain a set of fluorescent standards 1 - 4 (50 µL each loaded in triplicate) in addition to a single 50 µL real-time PCR reaction
for each library requiring amplification.
In order to maintain optimal library diversity it is necessary to add sufficient adaptor-ligated library DNA to each enrichment PCR reaction. The
optimal cycle number is dependant on the volume and concentration of library material added to each 50 uL PCR reaction. High background
fluorescence may result if >100 ng dsDNA template is added per 50 µL real-time PCR reaction.
Refer to Table 1 (page 12) for the suggested reaction setup for specific library preparation protocols.
Fluorescent standard set up requires the addition of 50 µL of each fluorescent standard (1 - 4) in triplicate to wells of the real-time PCR plate.
Change tips after each pipetting step. Seal each reaction, mix gently and centrifuge briefly.
5.5 Cycling protocol
Refer to Table 1 for the thermal cycling protocol for specific library types.
If conventional end-point PCR has previously been used successfully and the same amount and type of library is added to the KAPA HiFi
HotStart Real-Time PCR reactions, then program the real-time thermocycler with the same number of cycles as previously used. If the
optimal cycle number is not known refer to section 5.7 for more information.
5.6 Clean up PCR
After enrichment PCR, clean up each reaction using either Agencourt AMPure XP beads (Beckman Coulter Genomics part # A63881) or
Qiagen MinElute PCR Purification Kit (Qiagen, part # 28004).
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
5. Real-Time Library Amplification (cont.)
5.7 Validate library
Initially, the raw data (i.e., not background subtracted) linear real-time amplification plots can be used as a built-in quality metric
to validate the level of amplification of each amplified library.
If the linear amplification profile of the library is significantly below fluorescent standard 1 at the end of qPCR cycling, then it
is unlikely that there will be sufficient library material to sequence after PCR purification.
If the linear amplification profile of the library is significantly above fluorescent standard 3 at the end of qPCR cycling, then
the library has been over-amplified. This may lead to 1) amplification bias, 2) higher error rates, and/or 3) the presence of
chimeric PCR products.
This data is also useful as a quality control metric for identifying inconsistencies during library preparation between multiple
libraries.
NOTE: The amplification plots can also be used in real-time to select the optimal cycle without a pre-programmed termination
cycle. To do this:
1. Program 30 cycles into the real-time thermocycler.
2. After starting the real-time thermocycler, wait until the desired fluorescence of the library is achieved before terminating the
real-time reaction.
NOTE: It is critical to terminate the reaction directly after data acquisition at 72ºC and before the tube ramps to 95ºC for the start
of the next cycle. This will ensure that the enriched library DNA remains double-stranded for efficient downstream purification.
To verify the size of the PCR enriched fragments, check the size distribution by performing gel electrophoresis.
5.8 Library Quantification
Accurate quantification of amplifiable library molecules is critical for the efficient use of next-generation sequencing platforms.
Overestimation of library concentration results in lower cluster density after bridge PCR. Underestimation of library concentration
results in too many clusters on the flow cell, which can lead to poor cluster resolution. Both scenarios result in suboptimal
sequencing capacity. Accurate library quantification is equally important when pooling indexed libraries for multiplexed
sequencing to ensure equal representation of each library.
Integrate KAPA HiFi Real-Time PCR Library Amplification Kit with the appropriate KAPA Library Quantification Kit (KK4824,
KK4835, KK4844, KK4854) to accurately quantify the number of PCR-competent molecules. If libraries have been terminated
between fluorescent standards 1 – 3, a single 1:1,000 dilution of each library will be required for library quantification using the
KAPA Library Quantification Kits.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
5. Real-Time Library Amplification (cont.)
Table 1. Recommended reaction setup and cycling parameters for KAPA HiFi HotStart Real-Time PCR Master Mix (2X) reactions:
Library
Component
Final Conc.
PCR grade water
Genomic DNA
2X KAPA HiFi HS RT-PCR MM
PCR Primer 1.1
PCR Primer 2.1
ChIP
Volume/50 µL rxn
As needed
1X
25 µL
500 nM
500 nM
1 µL
1 µL
Library DNA
As needed
PCR grade water
As needed
2X KAPA HiFi HS RT-PCR MM
Paired-End
PE PCR Primer 1.0
PE PCR Primer 2.0
25 µL
500 nM
500 nM
1 µL
1 µL
Cycling*
15 sec at 98 ºC
30 sec at 65 ºC
30 sec at 72 ºC
Final Extension
1 min at 72 ºC
Denaturation
45 sec at 98 ºC
Cycling*
15 sec at 98 ºC
30 sec at 65 ºC
30 sec at 72 ºC
Final Extension
1 min at 72 ºC
PCR grade water
As needed
Denaturation
45 sec at 98 ºC
Cycling*
15 sec at 98 ºC
30 sec at 65 ºC
30 sec at 72 ºC
PE PCR Primer InPE 1.0
PE PCR Primer InPE 2.0
PCR Primer Index 1 - 12
2X KAPA HiFi HS RT-PCR MM
TruSeq DNA
45 sec at 98 ºC
As needed
1X
500 nM
10 nM
500 nM
Library DNA
™
Denaturation
Library DNA
2X KAPA HiFi HS RT-PCR MM
Paired-End
Multiplex
1X
Cycling Protocol
PCR Primer Cocktail (PPC)
Library DNA
25 µL
1 µL
1 µL
1 µL
As needed
1X
500 nM each
25 µL
Final Extension
1 min at 72 ºC
Denaturation
45 sec at 98 ºC
Cycling*
15 sec at 98 ºC
30 sec at 60 ºC
30 sec at 72 ºC
5 µL
20 µL
Final Extension
1 min at 72 ºC
* The optimal cycling number will depend upon the volume and concentration of adaptor-ligated, size separated, purified
library DNA added to each enrichment PCR reaction. Typically this is in the 10-18 cycle range but may require optimization.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
Appendix A: Composition of Materials
10X KAPA End Repair Buffer - contains dNTPs and ATP
500 mM Tris-HCl
100 mM MgCl2
100 mM DTT
10 mM ATP
4 mM dATP
4 mM dCTP
4 mM dGTP
4 mM dTTP
pH 7.5 @ 25 °C
KAPA End Repair Enzyme Mix
3,000 U/mL T4 DNA Polymerase
10,000 U/mL T4 Polynucleotide Kinase
Supplied in: 100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% Glycerol, pH7.4 @ 25 °C
10x KAPA A-Tailing Buffer - contains dATP
100 mM Tris-HCl
100 mM MgCl2
500 mM NaCl
10 mM DTT
2 mM dATP
pH 7.9 @ 25 °C
KAPA A-Tailing Enzyme
5,000 U/mL (0.5 mg/mL) Klenow Fragment (3’ ➞ 5’ exo-)
Supplied in: 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C
5X KAPA Ligation Buffer
330 mM Tris-HCl
50 mM MgCl2
5 mM DTT
5 mM ATP
30% PEG 6000
pH 7.6 @ 25 °C
KAPA DNA Ligase
600,000 U/mL (2 mg/mL) T4 DNA Ligase
Supplied in: 10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C
KAPA HiFi HotStart Real-Time PCR Master Mix
KAPA HiFi HotStart DNA Polymerase is an antibody-based hot start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA
polymerase engineered for increased processivity and high fidelity. KAPA HiFi HotStart DNA Polymerase has 5’ ➞ 3’ polymerase and
3’➞ 5’ exonuclease (proofreading) activities, but no 5’ ➞ 3’ exonuclease activity. The strong 3’ ➞ 5’ exonuclease activity results in superior
accuracy during DNA amplification. The error rate of KAPA HiFi HotStart DNA Polymerase is calculated at 1 error in 3.54 x 106 bases
covered (2.82 x 10-7). The SYBR® Green I intercalating dye contained within the master mix does not affect the fildelity of the polymerase.
DNA fragments generated with KAPA HiFi HotStart ReadyMix may be used for routine downstream analyses or applications, including
restriction enzyme digestion and sequencing. PCR products generated with KAPA HiFi HotStart ReadyMix are blunt-ended, but may be
3’-dA-tailed for cloning into TA cloning vectors.
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KAPA Library Preparation Kit
with Real-Time PCR Library Amplification
6. Storage, handling and specifications
6.1 Shipping, storage and handling
KAPA Library Preparation Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, store the entire kit at
-20 °C in a constant-temperature freezer. When stored under these conditions and handled correctly, all kit components will retain full activity
until the expiry date indicated on the kit.
Please note that certain components in KAPA Library Preparation Kits (e.g. End Repair Mix, DNA Ligase, buffers containing dNTPs and/or ATP,
etc.) are particularly sensitive to temperature and freeze-thaw cycles, and should be handled with special care. Minimize exposure of the KAPA
HiFi HotStart Real-Time PCR Master Mix (2X) and Flourescent Standards 1 - 4 to direct light. Exposure to direct light for an extended period of
time may result in loss of fluorescent signal intensity.
The KAPA HiFi HotStart Real-Time PCR Master Mix (2X) contains isostabilizers and may not freeze solidly, even when stored at -20 °C. Nevertheless,
always ensure that the KAPA HiFi HotStart Real-Time PCR Master Mix is fully thawed and has been vortexed before use.
KAPA HiFi HotStart Real-Time PCR Master Mix (2X) may be stored at 4 °C for regular, short-term use (up to 1 month). Long-term storage at room
temperature or 4 °C is not recommended. Please note that reagents stored above -20 °C are more prone to degradation when contaminated by
the user; storage at such temperatures is therefore at the user’s own risk.
6.2 Quality control
All kit components are subjected to stringent quality control tests, are free of contaminating exo- and endonuclease activities and meet strict
requirements with respect to DNA contamination. Detailed quality control information for individual kit components is available upon request,
please contact support@kapabiosystems.com.
6.3 Product use limitations and licenses
KAPA Library Preparation Kits are developed, designed and sold exclusively for research purposes and in vitro use. Neither the product, nor any individual component, has been tested
for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to the MSDS, which is available on request.
Certain applications of this product are covered by patents issued to parties other than Kapa Biosystems and applicable in certain countries. Purchase of this product does not include
a license to perform any such applications. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which
the product is used.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product
includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right
under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation
reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only.
Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing,
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
This product is provided under an agreement between Molecular Probes, Inc. and Kapa Biosystems Inc., and the manufacture, use, sale or import of this product is subject to one
or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Molecular Probes, Inc. The purchase of this product conveys to the buyer
the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include
testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components
or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components
for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in
manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic
purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this
product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851. SYBR® is a registered trademark of Molecular Probes, Inc.
The purchase of this product includes a limited, non-transferable license under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah
Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to
the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and
5,871,908, other than for the amount of product contained herein.
Licensed under U.S. Patent nos. 5,338,671 and 5,587,287 and corresponding patents in other countries. TruSeq is a trademark of Illumina, Inc. SYBR® is a registered trademark of
Molecular Probes, Inc.
For technical support please contact support@kapabiosystems.com
Boston, Massachusetts, United States
600 West Cummings Park, Suite 2250
Woburn, MA 01801 U.S.A.
Tel: +1 781 497 2933 Fax: +1 781 497 2934
Cape Town, South Africa
2nd Floor, Old Warehouse Building, Black River Park,
Fir Road, Observatory 7925, Cape Town, South Africa
Tel: +27 21 448 8200 Fax: +27 21 448 6503
www.kapabiosystems.com
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Email: info@kapabiosystems.com
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