Nextera XT DNA Library Preparation Experienced User Card (15031943 D)

Nextera XT DNA Library Preparation Experienced User Card (15031943 D)
Nextera XT Library Preparation
Experienced User Card
FOR RESEARCH USE ONLY
Date: __________________________
Illumina Kit Lot #: __________________________
Description: ______________________________________
_________________________________________________
NOTE
New or less experienced users are advised to follow the protocol in the Nextera XT DNA Library Preparation
Guide (part # 15031943) before using this document to perform the protocol.
ILLUMINA PROPRIETARY
Catalog # FC-131-9001DOC
Part # 15031943 Rev. D
September 2014
Page 1 of 18
Consumables
Item
Lot Number
2N NaOH (HP3)
Lot #: _____________________
Elute Target Buffer 2 (ET2)
Lot #: _____________________
Enrichment Elution Buffer 1 (EE1)
Lot #: _____________________
Enrichment Hybridization Buffer (EHB)
Lot #: _____________________
Enrichment Wash Solution (EWS)
Lot #: _____________________
Nextera Enrichment Amplification Mix (NEM)
Lot #: _____________________
Nextera Library Amplification Mix (NLM)
Lot #: _____________________
PCR Primer Cocktail (PPC)
Lot #: _____________________
Resuspension Buffer (RSB)
Lot #: _____________________
Sample Purification Beads (SPB)
Lot #: _____________________
Stop Tagment Buffer (ST)
Lot #: _____________________
Streptavidin Magnetic Beads (SMB)
Lot #: _____________________
Tagment DNA Buffer (TD)
Lot #: _____________________
Tagment DNA Enzyme (TDE1)
Lot #: _____________________
80% Ethanol
Lot #: _____________________
Page 2 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Index 1 Primers
Lot Number
Index Primer N701
Lot #: _____________________
Index Primer N702
Lot #: _____________________
Index Primer N703
Lot #: _____________________
Index Primer N704
Lot #: _____________________
Index Primer N705
Lot #: _____________________
Index Primer N706
Lot #: _____________________
Index Primer N707
Lot #: _____________________
Index Primer N708
Lot #: _____________________
Index Primer N709
Lot #: _____________________
Index Primer N710
Lot #: _____________________
Index Primer N711
Lot #: _____________________
Index Primer N712
Lot #: _____________________
Index Primer N714
Lot #: _____________________
Index Primer N715
Lot #: _____________________
Index Primer N716
Lot #: _____________________
Index Primer N717
Lot #: _____________________
Index Primer N718
Lot #: _____________________
Index Primer N719
Lot #: _____________________
Index Primer N720
Lot #: _____________________
Index Primer N721
Lot #: _____________________
Index Primer N722
Lot #: _____________________
Index Primer N723
Lot #: _____________________
Index Primer N724
Lot #: _____________________
Index Primer N726
Lot #: _____________________
Index Primer N727
Lot #: _____________________
Index Primer N728
Lot #: _____________________
Index Primer N729
Lot #: _____________________
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 3 of 18
Index 2 Primers
Lot Number
Index Primer S502
Lot #: _____________________
Index Primer S503
Lot #: _____________________
Index Primer S504
Lot #: _____________________
Index Primer S505
Lot #: _____________________
Index Primer S506
Lot #: _____________________
Index Primer S507
Lot #: _____________________
Index Primer S508
Lot #: _____________________
Index Primer S510
Lot #: _____________________
Index Primer S511
Lot #: _____________________
Index Primer S513
Lot #: _____________________
Index Primer S515
Lot #: _____________________
Index Primer S516
Lot #: _____________________
Index Primer S517
Lot #: _____________________
Index Primer S518
Lot #: _____________________
Index Primer S519
Lot #: _____________________
Index Primer S520
Lot #: _____________________
Index Primer S521
Lot #: _____________________
Index Primer S522
Lot #: _____________________
Page 4 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Tagmentation of Input DNA
During this step, input DNA is tagmented (tagged and fragmented) by the Nextera XT
transposome. The Nextera XT transposome simultaneously fragments the input DNA and adds
adapter sequences to the ends, allowing amplification by PCR in subsequent steps.
Estimated Time (8 reactions)
} Hands-on: 7 minutes
} Total duration: 17 minutes
Consumables
Item
Quantity
Storage
Supplied By
ATM (Amplicon Tagment Mix)
1 tube
-25°C to -15°C
Illumina
TD (Tagment DNA Buffer)
1 tube
-25°C to -15°C
Illumina
NT (Neutralize Tagment Buffer)
1 tube
Room
temperature
Illumina
-25°C to -15°C
User
Input DNA (0.2 ng/µl)
96-well hard shell TCY plate
1 plate
Microseal 'B' adhesive film
User
User
Preparation
[_] 1
Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice.
[_] 2
Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex
until all particulates are resuspended.
[_] 3
After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin
in a microcentrifuge.
Make NTA
NOTE
Make sure that the reaction is assembled in the order described for optimal kit performance.
You do not need to assemble the reaction on ice.
[_] 1
Label a new 96-well TCY plate NTA (Nextera XT Tagment Amplicon Plate).
[_] 2
Add 10 µl TD Buffer to each well to be used in this assay. Change tips between samples.
NOTE
Calculate the total volume of TD for all reactions, and divide the volume equally among the
wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA plate.
[_] 3
Add 5 µl input DNA at 0.2 ng/µl (1 ng total) to each sample well of the NTA plate.
[_] 4
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
[_] 5
Add 5 µl ATM to the wells containing input DNA and TD Buffer. Change tips between
samples.
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 5 of 18
NOTE
Calculate the total volume of ATM for all reactions, and divide the volume equally among the
wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA plate.
[_] 6
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
[_] 7
Seal the NTA plate with a Microseal 'B' adhesive seal.
[_] 8
Centrifuge at 280 × g at 20°C for 1 minute.
[_] 9
Place the NTA plate in a thermal cycler and run the following program:
NOTE
Make sure that the thermal cycler lid is heated during the incubation.
• 55°C for 5 minutes
Start time: _____________________
Stop time: _____________________
• Hold at 10°C
[_] 10 When the sample reaches 10°C, proceed immediately to Neutralize NTA as the transposome
is still active.
Neutralize NTA
NOTE
Calculate the total volume of NT for all reactions, and divide the volume equally among the
wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA plate.
[_] 1
Carefully remove the Microseal 'B' seal and add 5 µl NT Buffer to each well of the NTA
plate. Change tips between samples.
[_] 2
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples.
[_] 3
Seal the NTA plate with a Microseal 'B' adhesive seal.
[_] 4
Centrifuge at 280 × g at 20°C for 1 minute.
[_] 5
Place the NTA plate at room temperature for 5 minutes.
[_] 6
[Optional] Assess tagmentation by running 1 µl of sample on an HS bioanalyzer chip.
Start time: _____________________
Stop time: _____________________
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 6 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
PCR Amplification
In this step, the tagmented DNA is amplified via a limited-cycle PCR program. The PCR step
also adds index 1 (i7) and index 2 (i5) and sequences required for cluster formation. It is critical
to use the full amount of recommended input DNA and not add extra cycles of PCR cycles to
ensure libraries that produce high-quality sequencing results.
Estimated Time (8 reactions)
} Hands-on: 7 minutes
} Cycle time: 38 minutes
} Total duration: 45 minutes
Consumables
Item
Quantity
Storage
Supplied By
NPM (Nextera PCR Master Mix)
1 tube
-25°C to -15°C
Illumina
Index 1 primers (N7XX)
1 tube each index
-25°C to -15°C
Illumina
Index 2 primers (S5XX)
1 tube each index
-25°C to -15°C
Illumina
TruSeq Index Plate Fixture
Illumina
Microseal 'A' film
User
Preparation
[_] 1
If preparing the full set of 24/96 libraries for pooling and sequencing, proceed to step 2. If
less than a full set of libraries is pooled for sequencing, make sure that the correct index 1
(i7) and index 2 (i5) primers have been selected. Use the Illumina Experiment Manager and
the Nextera XT DNA Library Preparation Low-Plex Pooling Guidelines Tech Note to confirm the
selected index primers.
[_] 2
Remove NPM and the index primers (i5 and i7) from -25°C to -15°C storage and thaw on a
bench at room temperature.
Allow approximately 20 minutes to thaw NPM and index primers.
Start time: _____________________
[_] 3
Stop time: _____________________
After all reagents are thawed, gently invert each tube 3–5 times to mix and briefly centrifuge
the tubes in a microcentrifuge. Use 1.7 ml Eppendorf tubes as adapters for the
microcentrifuge.
[_] 4
For 24 libraries arrange the index primers in the TruSeq Index Plate Fixture using the
following arrangement:
[_] a Arrange index 1 (i7) primers (orange caps) in order horizontally.
[_] b Arrange index 2 (i5) primers (white caps) in order vertically.
[_] c To avoid index cross-contamination, discard the original caps and apply new caps
provided in the kit.
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 7 of 18
[_] 5
For 96 libraries arrange the index primers in the TruSeq Index Plate Fixture using the
following arrangement:
[_] a Arrange index 1 (i7) primer tubes (orange caps) in order horizontally.
[_] b Arrange index 2 (i5) primers (white caps) in order vertically.
[_] c To avoid index cross-contamination, discard the original caps and apply new caps
provided in the kit.
Amplify NTA
[_] 1
Place the NTA plate in the TruSeq Index Plate Fixture.
[_] 2
Add 15 µl NPM to each well of the NTA plate containing index primers. Change tips
between samples.
NOTE
Calculate the total volume of NPM for all reactions, and divide the volume equally among the
wells of a PCR eight-tube strip. Use a multichannel pipette to dispense into the NTA plate.
[_] 3
Using a multichannel pipette, add 5 µl index 2 primers (white caps) to each column of the
NTA plate. Changing tips between columns is required to avoid cross-contamination.
[_] 4
Using a multichannel pipette, add 5 µl index 1 primers (orange caps) to each row of the
NTA plate. Tips must be changed after each row to avoid index cross-contamination.
[_] 5
To avoid index cross-contamination, discard the original white caps and apply new white
caps provided in the kit.
[_] 6
To avoid index cross-contamination, discard the original orange caps and apply new orange
caps provided in the kit. Remove all the index primer tubes from the working area.
[_] 7
Using a multichannel pipette, gently pipette up and down 5 times to mix. Change tips
between samples to avoid index and sample cross-contamination.
[_] 8
Cover the NTA plate with Microseal 'A' film and seal with a rubber roller.
[_] 9
Centrifuge the NTA plate at 280 × g at 20°C for 1 minute.
[_] 10 Perform PCR using the following program on a thermal cycler:
NOTE
Make sure that the thermal cycler lid is heated during the incubation.
• 72°C for 3 minutes
Start time: _____________________
Stop time: _____________________
• 95°C for 30 seconds
• 12 cycles of:
— 95°C for 10 seconds
— 55°C for 30 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
Start time: _____________________
Stop time: _____________________
• Hold at 10°C
SAFE STOPPING POINT
If you do not plan to proceed immediately to PCR Clean-Up, there are two options for storage.
The NTA plate can remain on the thermal cycler overnight or you can store it at 2°C to 8°C
for up to two days.
Page 8 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
PCR Clean-Up
This step uses AMPure XP beads to purify the library DNA, and provides a size selection step
that removes short library fragments from the population.
Estimated Time (8 reactions)
} Hands-on: 15 minutes
} Total duration: 30 minutes
Consumables
Item
Quantity
Storage
Supplied By
RSB (Resuspension Buffer)
1 tube
-25°C to -15°C
Illumina
2°C to 8°C
User
AMPure XP beads
Freshly prepared 80% ethanol
(EtOH)
User
96-well MIDI plates
1 plate
User
96-well TCY plates
1 plate
User
Preparation
[_] 1
Bring the AMPure XP beads to room temperature.
[_] 2
Prepare fresh 80% ethanol from absolute ethanol.
NOTE
Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air
impacting your results.
Make CAN
[_] 1
Centrifuge the NTA plate at 280 × g for 1 min (20˚C) to collect condensation.
[_] 2
Label a new MIDI plate CAA (Clean Amplified Plate).
[_] 3
Using a multichannel pipette set to 50 µl, transfer the PCR product from the NTA plate to
the CAA plate. Change tips between samples.
[_] 4
Vortex the AMPure XP beads for 30 seconds to make sure that the beads are evenly
dispersed. Add an appropriate volume of beads to a trough.
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 9 of 18
[_] 5
Using a multichannel pipette, add 30 µl AMPure XP beads to each well of the CAA plate.
For 2x250 or 2x300 runs on the MiSeq, add 25 µl of AMPure XP beads to each well of the
CAA plate.
Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To
maximize recovery of smaller fragments out of the SPRI cleanup, Illumina recommends the
following conditions:
Size of Largest Amplicon in
Pool
AMPure XP Recommendation
AMPure XP Volume
< 300 bp
1.8x AMPure XP*
90 µl
300–500 bp
1.8x AMPure XP
90 µl
> 500 bp
0.6x AMPure XP
(0.5x AMPure XP for 2x250
runs on the MiSeq)
30 µl
(25 µl for 2x250 or 2x300 runs
on the MiSeq)
* Illumina recommends > 300 bp to ensure even coverage across the length of the DNA fragment. An expected
drop off in sequencing coverage about 50 bp from each distal end of a fragment can be seen. The drop off is
because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. This enzymatic
clipping of PCR primers avoids wasted sequencing output on non-informative bases that do not contain
genomic inserts. If you wish to sequence the genomic loci contained within a PCR primer, simply design
your amplicons to be ~100 bases larger than the desired insert to be sequenced.
[_] 6
Gently pipette mix up and down 10 times.
NOTE
Alternatively the solution can be mixed by shaking the CAA plate on a microplate shaker at
1800 rpm for 2 minutes.
[_] 7
Incubate at room temperature without shaking for 5 minutes.
Start time: _____________________
[_] 8
Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.
Start time: _____________________
[_] 9
Stop time: _____________________
Stop time: _____________________
With the CAA plate on the magnetic stand, use a multichannel pipette to remove and
discard the supernatant carefully. Change tips between samples.
NOTE
If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate.
Then let the plate rest on the magnet for 2 minutes and confirm that the supernatant has
cleared.
[_] 10 With the CAA plate on the magnetic stand, wash the beads with freshly prepared 80%
ethanol as follows:
[_] a Using a multichannel pipette, add 200 µl freshly prepared 80% ethanol to each sample
well. Do not resuspend the beads yet.
[_] b Incubate the plate on the magnetic stand for 30 seconds.
[_] c Carefully remove and discard the supernatant.
[_] 11 With the CAA plate on the magnetic stand, perform a second ethanol wash as follows:
[_] a Using a multichannel pipette, add 200 µl freshly prepared 80% ethanol to each sample
well.
[_] b Incubate the plate on the magnetic stand for 30 seconds.
[_] c Carefully remove and discard the supernatant.
[_] d Use a P20 multichannel pipette with fine pipette tips to remove excess ethanol.
Page 10 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
[_] 12 With the CAA plate still on the magnetic stand, allow the beads to air-dry for 15 minutes.
Start time: _____________________
Stop time: _____________________
[_] 13 Remove the CAA plate from the magnetic stand. Using a multichannel pipette, add 52.5 µl
RSB to each well of the CAA plate.
[_] 14 Gently pipette mix up and down 10 times, changing tips after each column.
NOTE
Alternatively the solution can be mixed by shaking the CAA plate on a microplate shaker at
1800 rpm for 2 minutes.
[_] 15 Incubate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 16 Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.
Start time: _____________________
Stop time: _____________________
[_] 17 Label a new TCY plate CAN (Clean Amplified NTA Plate).
[_] 18 Using a multichannel pipette, carefully transfer 50 µl of the supernatant from the CAA plate
to the CAN plate. Change tips between samples to avoid cross-contamination.
SAFE STOPPING POINT
If you do not plan to proceed to Library Normalization immediately, seal the CAN plate with
Microseal “B” adhesive seal and store it at -25°C to -15°C for up to a week.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 11 of 18
Page 12 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Library Normalization
This process normalizes the quantity of each library to ensure more equal library representation
in your pooled sample.
Estimated Time (96 reactions)
} Total duration: 1 hour 20 minutes
} Hands-on: 30 minutes
Consumables
Item
Quantity
Storage
Supplied By
LNA1 (Library Normalization
Additives 1)
1 tube
-25°C to -15°C
Illumina
LNB1 (Library Normalization
Beads 1)
1 tube
2°C to 8°C
Illumina
LNW1 (Library Normalization
Wash 1)
2 tubes
2°C to 8°C
Illumina
LNS1 (Library Normalization
Storage Buffer 1)
1 tube
Room
temperature
Illumina
0.1 N NaOH (less than one week
old)
3 ml per 96 samples
User
96-well MIDI plate
1 plate
User
96-well TCY plate
1 plate
User
15 ml conical tube
1 tube
User
Preparation
NOTE
Illumina recommends performing the LNA1 preparation step under a fume hood.
[_] 1
Remove LNA1 from -25°C to -15°C storage and bring to room temperature. Use a 20°C to
25°C water bath as needed. When at room temperature, vortex vigorously to make sure that
all precipitates have dissolved.
[_] 2
Remove LNB1 and LNW1 from 2°C to 8°C storage and bring to room temperature.
[_] 3
Vigorously vortex LNB1 for at least 1 minute with intermittent inversion. Repeat this step
until the beads are well-resuspended and no pellet is found at the bottom of the tube when
the tube is inverted.
[_] 4
Make sure that LNS1 is at room temperature before use.
Elute LNP
[_] 1
Label a new MIDI plate LNP (Library Normalization Plate).
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 13 of 18
[_] 2
Using a P20 multichannel pipette and fine tips, carefully transfer 20 µl of the supernatant
from the CAN plate to the LNP plate. Change tips between samples to avoid crosscontamination.
[_] 3
For 96 samples, add 4.4 ml LNA1 to a fresh 15 ml conical tube.
[_] 4
Use a P1000 pipette set to 1000 µl to resuspend LNB1 thoroughly. Pipette up and down 15–
20 times, until the bead pellet at the bottom is resuspended.
WARNING
It is critical to resuspend the LNB1 bead pellet at the bottom of the tube completely. The use of
a P1000 ensures that the beads are homogeneously resuspended and that there is no bead
mass at the bottom of the tube. This resuspension is essential for achieving consistent cluster
density on the flow cell.
[_] 5
Immediately after LNB1 is thoroughly resuspended, use a P1000 pipette to transfer 800 µl
LNB1 to the 15 ml conical tube containing LNA1. Mix well by inverting the tube 15–20
times. The resulting LNA1/LNB1 bead mix is enough for 96 samples. Pour the bead mix into
a trough and use it immediately in the next step.
NOTE
If you do not plan to use full tubes for 96 samples, a P1000 set to 1000 µl is still required to
resuspend the beads completely in step 4. Mix only the required amounts of LNA1 and LNB1
for the current experiment. Never use a P200 pipette to handle LNB1. Store the remaining
LNA1 and LNB1 separately at their respective recommended temperatures. If not used
immediately, never freeze or mix the LNB1 beads with LNA1 to preserve stability.
[_] 6
Using a multichannel pipette, add 45 µl combined LNA1/LNB1 to each well of the LNP
plate containing libraries.
[_] 7
Seal the LNP plate with a Microseal 'B' adhesive seal and shake on a microplate shaker at
1800 rpm for 30 minutes.
[_] 8
Start time: _____________________
Stop time: _____________________
Start time: _____________________
Stop time: _____________________
Place the plate on a magnetic stand for 2 minutes and confirm that the supernatant has
cleared.
Start time: _____________________
[_] 9
Stop time: _____________________
With the LNP plate on the magnetic stand, use a multichannel pipette set to 80 µl to remove
the supernatant and then discard in an appropriate hazardous waste container.
[_] 10 Remove the LNP plate from the magnetic stand and wash the beads with LNW1, as follows:
[_] a Using a multichannel pipette, add 45 µl LNW1 to each sample well.
[_] b Seal the LNP plate with a Microseal 'B' adhesive seal.
[_] c Shake the LNP plate on a microplate shaker at 1800 rpm for 5 minutes.
Start time: _____________________
[_] d
Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.
Start time: _____________________
[_] e
Stop time: _____________________
Stop time: _____________________
Carefully remove and discard the supernatant in an appropriate hazardous waste
container.
[_] 11 Repeat the LNW1 wash described in the previous step.
[_] 12 Remove the LNP plate from the magnetic stand and add 30 µl 0.1 N NaOH (fresh stock
solution) to each well to elute the sample.
Page 14 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
[_] 13 Seal the LNP plate with a Microseal 'B' adhesive seal and shake on a microplate shaker at
1800 rpm for 5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 14 During the 5 minute elution, apply the SGP (Storage Plate) barcode plate sticker to a new 96well PCR plate.
[_] 15 Add 30 µl LNS1 to each well to be used in the SGP plate.
[_] 16 After the 5 minute elution, make sure that all samples in the LNP plate are resuspended
completely. If the samples are not resuspended, gently pipette those samples up and down
or lightly tap the plate on the bench to resuspend the beads. Then shake for another 5
minutes.
[_] 17 Place the LNP plate on the magnetic stand for 2 minutes or until the liquid is clear.
[_] 18 Using a multichannel pipette set to 30 µl, transfer the supernatant from the LNP plate to the
SGP plate. Change tips between samples to avoid cross-contamination.
[_] 19 Seal the SGP plate with Microseal 'B' and then centrifuge to 1000 × g for 1 minute.
SAFE STOPPING POINT
If you do not plan to proceed to Library Pooling and MiSeq Sample Loading following the
completion of Library Normalization, store the sealed SGP plate at -25°C to -15°C for up to
one week.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 15 of 18
Page 16 of 18
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Library Pooling and MiSeq Sample Loading
In preparation for cluster generation and sequencing, equal volumes of normalized library are
combined, diluted in Hybridization Buffer, and heat denatured before sequencing.
Estimated Time (8 reactions)
} Total duration: 5 minutes
} Hands-on: 5 minutes
Estimated Time (24 reactions)
} Total duration: 10 minutes
} Hands-on: 10 minutes
Consumables
Item
Quantity
Storage
Supplied By
HT1 (Hybridization Buffer)
1 tube
-25°C to -15°C
Illumina
MiSeq reagent cartridge
1 cartridge
-25°C to -15°C
Illumina
Eppendorf LoBind
Microcentrifuge Tube
1 tube
User
PCR 8-tube strip
1
User
2.5 L Ice bucket
1
User
Preparation
[_] 1
Set a heat block suitable for 1.5 ml centrifuge tubes to 96°C.
[_] 2
Remove a MiSeq reagent cartridge from -25°C to -15°C storage and thaw at room
temperature.
[_] 3
In an ice bucket, prepare an ice-water bath by combining 3 parts ice and 1 part water.
Make DAL
NOTE
If you are clustering a HiSeq or GAIIx flow cell, see the Clustering Nextera XT Samples for the
HiSeq, HiScanSQ, and GAIIx section in the Nextera XT Sample Preparation Guide (part # 15031942)
for additional instructions.
[_] 1
If the SGP plate was stored frozen, thaw the plate at room temperature.
[_] 2
Centrifuge the SGP plate at 1000 × g for 1 minute at 20°C to collect condensation.
[_] 3
If the SGP plate was stored frozen, mix each library to be sequenced by pipetting up and
down 3–5 times using a P200 multichannel pipette. Change tips between samples.
[_] 4
Using a P20 multichannel pipette, transfer 5 µl of each library to be sequenced from the SGP
plate, column by column, to a PCR 8-tube strip. Change tips after each column to avoid
cross-contamination.
[_] 5
Label a fresh Eppendorf tube PAL (Pooled Amplicon Library).
Nextera XT Library Preparation
Experienced User Card
Part # 15031943 Rev. D
Page 17 of 18
[_] 6
Combine and transfer the contents of the PCR 8-tube strip into the PAL tube. Mix PAL well.
[_] 7
Label a fresh Eppendorf tube DAL (Diluted Amplicon Library).
[_] 8
Add 576 µl HT1 to the DAL tube.
[_] 9
Transfer 24 µl PAL to the DAL tube containing HT1. Using the same tip, pipette up and
down 3–5 times to rinse the tip and ensure complete transfer.
[_] 10 Mix DAL by vortexing the tube at top speed.
[_] 11 Using a heat block, incubate the DAL tube at 96°C for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 12 After the incubation, invert DAL 1–2 times to mix and immediately place in the ice-water
bath.
[_] 13 Keep the DAL tube in the ice-water bath for 5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 14 Load DAL into a thawed MiSeq reagent cartridge into the Load Samples reservoir.
[_] 15 Cap the PAL tube and seal the SGP plate with a Microseal ‘B’ adhesive seal.
[_] 16 Store the PAL tube and the SGP plate at -25°C to -15°C for up to one week.
[_] 17 Sequence your library as indicated in the MiSeq System User Guide (part # 15027617).
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
*15031943*
Part # 15031943 Rev. D
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