Nextera XT DNA Library Prep Checklist (1000000006566 v00)

Nextera XT DNA Library Prep Checklist (1000000006566 v00)
For Research Use Only. Not for
use in diagnostic procedures.
Nextera XT DNA
Tagment Genomic DNA
□1
Add the following to a new PCR plate. Pipette to
mix.
Item
TD
Normalized gDNA
□2
□3
□4
□5
□6
□7
□8
Volume (µl)
10
5
Add 5 µl ATM. Pipette to mix.
Centrifuge at 280 × g at 20°C for 1 minute.
Place on the thermal cycler and run the
tagmentation program.
Add 5 µl NT. Pipette to mix.
Centrifuge at 280 × g at 20°C for 1 minute.
Incubate at room temperature for 5 minutes.
[Optional] Run 1 µl sample on a High Sensitivity
DNA chip.
Amplify Libraries
□1
□2
□3
□4
□5
□6
□7
[24 libraries] Arrange the index primers as
follows.
} Arrange Index 1 (i7) adapters in columns 1–6.
} Arrange Index 2 (i5) adapter in rows A–D.
[96 libraries] Arrange the index primers as
follows.
} Arrange Index 1 (i7) adapters in columns 1–
12.
} Arrange Index 2 (i5) adapter in rows A–H.
Add 5 µl of each Index 1 (i7) adapter down each
column. Replace the cap on each i7 adapter tube
with a new orange cap.
Add 5 µl of each Index 2 (i5) adapter across each
row. Replace the cap on each i5 adapter tube
with a new white cap.
Add 15 µl NPM. Pipette to mix.
Centrifuge at 280 × g at 20°C for 1 minute.
Place on the thermal cycler and run the PCR
program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
2°C to 8°C for up to 2 days. Alternatively, leave on
the thermal cycler overnight.
Document # 1000000006566 v00
January 2016
ILLUMINA PROPRIETARY
Clean Up Libraries
□1
□2
□3
□4
□5
□6
□7
□8
□9
□10
□11
□12
□13
□14
□15
□16
Centrifuge at 280 × g at 20°C for 1 minute.
Transfer 50 µl PCR product.
Add 30 µl AMPure XP beads.
Shake at 1800 rpm for 2 minutes.
Incubate at room temperature for 5 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 200 µl 80% EtOH.
Using a 20 µl pipette, remove residual 80%
EtOH.
Air-dry on the magnetic stand for 15 minutes.
Remove from the magnetic stand.
Add 52.5 µl RSB.
Shake at 1800 rpm for 2 minutes.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand until liquid is clear.
Transfer 50 µl supernatant.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Page 1 of 3
For Research Use Only. Not for
use in diagnostic procedures.
Nextera XT DNA
Check Libraries
□1
[Optional] Run 1 µl of undiluted library on an
Agilent Technology 2100 Bioanalyzer using a
High Sensitivity DNA chip.
Normalize Libraries
□1
□2
□3
□4
□5
□6
□7
□8
□9
□10
□11
□12
□13
□14
□15
□16
□17
□18
Transfer 20 µl supernatant.
[96 samples] Add 4.4 ml LNA1 to a new 15 ml
conical tube.
Thoroughly resuspend LNB1. Pipette to mix.
Transfer 800 µl LNB1 to the tube. Invert to mix.
Add 45 µl combined LNA1/LNB1.
Shake at 1800 rpm for 30 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 45 µl LNW1.
Add 30 µl 0.1 N NaOH.
Shake at 1800 rpm for 5 minutes.
During the 5 minute elution, label a new 96-well
PCR plate SGP.
Add 30 µl LNS1 to the SGP plate. Set aside.
After the 5 minute elution, make sure that all
samples are resuspended. Pipette to mix.
Shake at 1800 rpm for 5 minutes.
Place on a magnetic stand until liquid is clear.
Transfer the supernatant from the midi plate to
the SGP plate.
Centrifuge at 1000 × g for 1 minute.
Pool Libraries
□1
□2
□3
□4
□5
□6
□7
□8
To prepare for the sequencing run, begin thawing
reagents according to the instructions for your
sequencing instrument.
If the SGP plate was stored frozen at -25°C to 15°C, thaw at room temperature. Pipette to mix.
Centrifuge at 1000 × g at 20°C for 1 minute.
Transfer 5 µl from the SGP plate to a new PCR 8tube strip.
Label a new Eppendorf tube PAL.
Transfer the contents of the PCR 8-tube strip to
the PAL tube. Invert to mix.
Dilute pooled libraries to the loading
concentration for the sequencing instrument you
are using. See the denature and dilute libraries
guide for your instrument.
Store unused pooled libraries in the PAL tube
and SGP plate at -25°C to -15°C for up to 7 days.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Page 2 of 3
January 2016
ILLUMINA PROPRIETARY
Document # 1000000006566 v00
Nextera XT DNA
For Research Use Only. Not for
use in diagnostic procedures.
Acronyms
Acronym
Definition
ATM
Amplicon Tagment Mix
CAA
Clean Amplified Plate
CAN
Clean Amplified NTA Plate
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNS1
Library Normalization Storage
Buffer 1
LNW1
Library Normalization Wash 1
LNP
Library Normalization Plate
NT
Neutralize Tagment Buffer
NPM
Nextera PCR Master Mix
NTA
Nextera XT Tagment Amplicon
Plate
PAL
Pooled Amplicon Library
RSB
Resuspension Buffer
SGP
Storage Plate
TD
Tagment DNA Buffer
Document # 1000000006566 v00
January 2016
ILLUMINA PROPRIETARY
Page 3 of 3
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