Nextera Rapid Capture Enrichment Checklist (15075702 v01)

Nextera Rapid Capture Enrichment Checklist (15075702 v01)
For Research Use Only. Not for
use in diagnostic procedures.
Nextera Rapid Capture Enrichment
Tagment Genomic DNA
□1
□2
□3
Quantify gDNA using a fluorometric method.
Dilute gDNA in Tris-HCl 10 mM, pH 8.5 to a
final volume of 10 μl at 5 ng/μl.
Add the following to the NLT plate.
Item
Normalized gDNA
TD
TDE1
□4
□5
□6
□
□
□
□
Volume (µl)
10
25
15
Shake at 1800 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on the 58°C microheating system with the
lid closed for 10 minutes.
7 Add 15 μl ST.
8 Shake at 1800 rpm for 1 minute.
9 Centrifuge at 280 × g for 1 minute.
10 Incubate at room temperature for 4 minutes.
Clean Up Tagmented DNA
□1
□2
□3
□4
□5
□6
□7
□8
□9
□10
□11
□12
□13
□14
□15
□16
Document # 15075702 v01
Add 65 μl SPB.
Shake at 1800 rpm for 1 minute.
Incubate at room temperature for 8 minutes.
Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until the 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 10 minutes.
Remove from the magnetic stand.
Add 22.5 μl RSB.
Shake at 1800 rpm for 1 minute.
Incubate at room temperature for 2 minutes.
Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until the liquid is
clear.
Transfer 20 μl supernatant to the NLA plate.
January 2016
ILLUMINA PROPRIETARY
Amplify Tagmented DNA
□1
□2
□3
□4
□5
□6
□7
□8
□9
Arrange Index 1 (i7) adapters in columns 1–12.
Arrange Index 2 (i5) adapters in rows A–H.
Place the plate on the TruSeq Index Plate Fixture.
Add 5 μl of each Index 1 adapter down each
column.
Add 5 μl of each Index 2 adapter across each
row.
Add 20 μl NLM.
Shake at 1200 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on the thermal cycler and run the NLM
AMP 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.
Page 1 of 5
For Research Use Only. Not for
use in diagnostic procedures.
Nextera Rapid Capture Enrichment
Clean Up Amplified DNA
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer 50 μl supernatant to the NLC plate.
□3 Add 90 μl SPB.
□4 Shake at 1800 rpm for 1 minute.
□5 Incubate at room temperature for 10 minutes.
□6 Centrifuge at 280 × g for 1 minute.
□7 Place on a magnetic stand until liquid is clear.
□8 Remove and discard all supernatant.
□9 Wash 2 times with 200 μl 80% EtOH.
□10 Using a 20 μl pipette, remove residual
□11
□12
□13
□14
□15
□16
□17
□18
80% EtOH.
Air-dry on the magnetic stand for 10 minutes.
Add 27 μl RSB.
Shake at 1800 rpm for 1 minute.
Incubate at room temperature for 2 minutes.
Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Transfer 25 μl supernatant to the NIL plate.
Quantify the library using a fluorometric method.
Hybridize Probes
□1
□2
Combine 500 ng of each DNA library. Make sure
that each library has a unique index.
} If the total volume is > 40 μl, concentrate the
pooled sample to 40 μl.
} If the total volume is < 40 μl, increase the
volume to 40 μl with RSB.
Add the following to the NEH1 plate.
Item
DNA library sample or pool
EHB
CEX, EEX, or RCO
□3
□4
□5
□6
Capture Hybridized Probes
Volume (µl)
40
50
10
Shake at 1200 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on the thermal cycler and run the NRC
HYB program.
Keep at the 58°C holding temperature for at least
90 minutes and up to 24 hours.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 14 days.
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer all to the NEW1 plate.
□3 Add 250 μl SMB.
□4 Shake at 1200 rpm for 5 minutes.
□5 Incubate at room temperature for 25 minutes.
□6 Centrifuge at 280 × g for 1 minute.
□7 Place on a magnetic stand until liquid is clear.
□8 Remove and discard all supernatant.
□9 Remove from the magnetic stand.
□10 Wash 2 times with 200 μl EWS.
□11 Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex
to mix.
□12 Add 23 μl elution premix.
□13 Shake at 1800 rpm for 2 minutes.
□14 Incubate at room temperature for 2 minutes.
□15 Centrifuge at 280 × g for 1 minute.
□16 Place on a magnetic stand until liquid is clear.
□17 Transfer 21 μl supernatant to the NEH2 plate.
□18 Add 4 μl ET2.
□19 Shake at 1200 rpm for 1 minute.
□20 Centrifuge at 280 × g for 1 minute.
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 5
January 2016
ILLUMINA PROPRIETARY
Document # 15075702 v01
Nextera Rapid Capture Enrichment
Perform Second Hybridization
□1
Add the following.
Reagent
RSB
EHB
CEX, EEX, or RCO
□2
□3
□4
□5
Volume (µl)
15
50
10
Shake at 1200 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on the thermal cycler and run the NRC
HYB program.
Keep at the 58°C holding temperature for at least
14.5 hours and up to 24 hours.
Perform Second Capture
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer all supernatant to the NEW2 plate.
□3 Add 250 μl SMB.
□4 Shake at 1200 rpm for 5 minutes.
□5 Incubate at room temperature for 25 minutes.
□6 Centrifuge at 280 × g for 1 minute.
□7 Place on a magnetic stand until liquid is clear.
□8 Remove and discard all supernatant.
□9 Remove from the magnetic stand.
□10 Wash 2 times with 200 μl EWS.
□11 Mix 28.5 μl EE1 and 1.5 μl HP3, and then vortex
to mix.
□12 Add 23 μl elution premix.
□13 Shake at 1800 rpm for 2 minutes.
□14 Incubate at room temperature for 2 minutes.
□15 Centrifuge at 280 × g for 1 minute.
□16 Place on a magnetic stand until liquid is clear.
□17 Transfer 21 μl supernatant to the NEC1 plate.
□18 Add 4 μl ET2.
□19 Shake at 1800 rpm for 1 minute.
□20 Centrifuge at 280 × g for 1 minute.
Document # 15075702 v01
January 2016
ILLUMINA PROPRIETARY
For Research Use Only. Not for
use in diagnostic procedures.
Clean Up Captured Library
□1 Add 45 μl SPB.
□2 Shake at 1800 rpm for 1 minute.
□3 Incubate at room temperature for 10 minutes.
□4 Centrifuge at 280 × g for 1 minute.
□5 Place on a magnetic stand until liquid is clear.
□6 Remove and discard all supernatant.
□7 Wash 2 times with 200 μl 80% EtOH.
□8 Use a 20 μl pipette to remove residual EtOH.
□9 Air-dry for 10 minutes.
□10 Remove from the magnetic stand.
□11 Add 27.5 μl RSB.
□12 Shake at 1800 rpm for 1 minute.
□13 Incubate at room temperature for 2 minutes.
□14 Centrifuge at 280 × g for 1 minute.
□15 Place on a magnetic stand until liquid is clear.
□16 Transfer 25 μl supernatant to the NEA plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Page 3 of 5
Nextera Rapid Capture Enrichment
Amplify Enriched Library
□1
□2
□3
□4
□5
Add 5 μl PPC.
Add 20 μl NEM.
Shake at 1200 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on the thermal cycler and run the AMP10
or AMP12 program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
2°C to 8°C for up to 2 days.
Clean Up Amplified Enriched
Library
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer 50 μl to the NEC2 plate.
□3 Add 90 μl SPB.
□4 Shake at 1800 rpm for 1 minute.
□5 Incubate at room temperature for 10 minutes.
□6 Centrifuge at 280 × g for 1 minute.
□7 Place on a magnetic stand until liquid is clear.
□8 Remove and discard all supernatant.
□9 Wash 2 times with 200 μl 80% EtOH.
□10 Use a 20 μl pipette to remove residual EtOH.
□11 Air-dry on the magnetic stand for 10 minutes.
□12 Remove from the magnetic stand.
□13 Add 32 μl RSB.
□14 Shake at 1800 rpm for 1 minute.
□15 Incubate at room temperature for 2 minutes.
□16 Centrifuge at 280 × g for 1 minute.
□17 Place on a magnetic stand until liquid is clear.
□18 Transfer 30 μl supernatant to the NEL plate.
For Research Use Only. Not for
use in diagnostic procedures.
Check Enriched Libraries
□1
□2
□3
Quantify using a fluorometric method.
If the concentration is higher than the
quantitative range for the High Sensitivity DNA
chip, dilute the library 1:10 with RSB.
Run 1 μl using a High Sensitivity DNA chip.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Page 4 of 5
January 2016
ILLUMINA PROPRIETARY
Document # 15075702 v01
Nextera Rapid Capture Enrichment
Acronyms
Acronym
Definition
Acronym
Definition
NLT
Nextera Library Tagment Plate
CEX
Coding Exome Oligos
PPC
PCR Primer Cocktail
EE1
Enrichment Elution Buffer 1
RCO
Rapid Capture Oligos
EEX
Expanded Exome Oligos
RSB
Resuspension Buffer
EHB
Enrichment Hybridization Buffer
SMB
Streptavidin Magnetic Beads
ET2
Elute Target Buffer 2
SPB
Sample Purification Beads
EWS
Enrichment Wash Solution
ST
Stop Tagment Buffer
HP3
2N NaOH
TD
Tagment DNA Buffer
NEA
Nextera Enrichment Amplification Plate
TDE1
Tagment DNA Enzyme TDE
NEC1
Nextera Enriched Clean Up Plate 1
NEC2
Nextera Enriched Clean Up Plate 2
NEH1
Nextera Enrichment Hyb Plate 1
NEH2
Nextera Enrichment Hyb Plate 2
NEL
Nextera Enrichment Library Plate
NEM
Enrichment Amp Mix
NEW1
Nextera Enrichment Wash Plate 1
NEW2
Nextera Enrichment Wash Plate 2
NIL
Nextera Index Library Plate
NLA
Nextera Library Amplification Plate
NLC
Nextera Library Clean Up Plate
NLM
Library Amp Mix
Document # 15075702 v01
January 2016
ILLUMINA PROPRIETARY
For Research Use Only. Not for
use in diagnostic procedures.
Page 5 of 5
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