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

2N NaOH (HP3)

Elute Target Buffer 2 (ET2)

Enrichment Elution Buffer 1 (EE1)

Enrichment Hybridization Buffer (EHB)

Enrichment Wash Solution (EWS)

Nextera Enrichment Amplification Mix (NEM)

Nextera Library Amplification Mix (NLM)

PCR Primer Cocktail (PPC)

Resuspension Buffer (RSB)

Sample Purification Beads (SPB)

Stop Tagment Buffer (ST)

Streptavidin Magnetic Beads (SMB)

Tagment DNA Buffer (TD)

Tagment DNA Enzyme (TDE1)

80% Ethanol

Lot Number

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

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Page 2 of 18

Nextera XT Library Preparation

Experienced User Card

Part # 15031943 Rev. D

Index 1 Primers

Index Primer N701

Index Primer N702

Index Primer N703

Index Primer N704

Index Primer N705

Index Primer N706

Index Primer N707

Index Primer N708

Index Primer N709

Index Primer N710

Index Primer N711

Index Primer N712

Index Primer N714

Index Primer N715

Index Primer N716

Index Primer N717

Index Primer N718

Index Primer N719

Index Primer N720

Index Primer N721

Index Primer N722

Index Primer N723

Index Primer N724

Index Primer N726

Index Primer N727

Index Primer N728

Index Primer N729

Nextera XT Library Preparation

Experienced User Card

Part # 15031943 Rev. D

Page 3 of 18

Lot Number

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

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Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

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Lot #: _____________________

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Index 2 Primers

Index Primer S502

Index Primer S503

Index Primer S504

Index Primer S505

Index Primer S506

Index Primer S507

Index Primer S508

Index Primer S510

Index Primer S511

Index Primer S513

Index Primer S515

Index Primer S516

Index Primer S517

Index Primer S518

Index Primer S519

Index Primer S520

Index Primer S521

Index Primer S522

Lot Number

Lot #: _____________________

Lot #: _____________________

Lot #: _____________________

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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

ATM (Amplicon Tagment Mix)

TD (Tagment DNA Buffer)

NT (Neutralize Tagment Buffer)

Quantity

1 tube

1 tube

1 tube

Storage

-25°C to -15°C

-25°C to -15°C

Room temperature

-25°C to -15°C

Supplied By

Illumina

Illumina

Illumina

Input DNA (0.2 ng/µl)

96-well hard shell TCY plate

Microseal 'B' adhesive film

1 plate

User

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

NPM (Nextera PCR Master Mix)

Index 1 primers (N7XX)

Index 2 primers (S5XX)

TruSeq Index Plate Fixture

Microseal 'A' film

Quantity

1 tube

1 tube each index

1 tube each index

Storage

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

Supplied By

Illumina

Illumina

Illumina

Illumina

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: _____________________ Stop time: _____________________

[_] 3 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

RSB (Resuspension Buffer)

AMPure XP beads

Freshly prepared 80% ethanol

(EtOH)

96-well MIDI plates

96-well TCY plates

Quantity

1 tube

1 plate

1 plate

Storage

-25°C to -15°C

2°C to 8°C

Supplied By

Illumina

User

User

User

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:

AMPure XP Recommendation AMPure XP Volume Size of Largest Amplicon in

Pool

< 300 bp

300–500 bp

> 500 bp

1.8x AMPure XP*

1.8x AMPure XP

0.6x AMPure XP

(0.5x AMPure XP for 2x250 runs on the MiSeq)

90 µl

90 µl

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: _____________________ Stop time: _____________________

[_] 8 Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.

Start time: _____________________ Stop time: _____________________

[_] 9 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

LNA1 (Library Normalization

Additives 1)

LNB1 (Library Normalization

Beads 1)

LNW1 (Library Normalization

Wash 1)

LNS1 (Library Normalization

Storage Buffer 1)

0.1 N NaOH (less than one week old)

96-well MIDI plate

96-well TCY plate

15 ml conical tube

Quantity

1 tube

1 tube

2 tubes

1 tube

3 ml per 96 samples

1 plate

1 plate

1 tube

Storage

-25°C to -15°C

2°C to 8°C

2°C to 8°C

Room temperature

Supplied By

Illumina

Illumina

Illumina

Illumina

User

User

User

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.

Start time: _____________________ Stop time: _____________________

Start time: _____________________ Stop time: _____________________

[_] 8 Place the plate on a magnetic stand for 2 minutes and confirm that the supernatant has cleared.

Start time: _____________________ Stop time: _____________________

[_] 9 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: _____________________ Stop time: _____________________

[_] d Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.

Start time: _____________________ Stop time: _____________________

[_] e 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

HT1 (Hybridization Buffer)

MiSeq reagent cartridge

Eppendorf LoBind

Microcentrifuge Tube

PCR 8-tube strip

2.5 L Ice bucket

1

1

Quantity

1 tube

1 cartridge

1 tube

Storage

-25°C to -15°C

-25°C to -15°C

Supplied By

Illumina

Illumina

User

User

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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