TruSight Rapid Capture Checklist (1000000005003 v00)

TruSight Rapid Capture Checklist (1000000005003 v00)
For Research Use Only. Not for
use in diagnostic procedures.
TruSight Rapid Capture Checklist
Tagment Genomic DNA
□1
□2
□
□
□
□
□
□
□
□
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.
3 Add the following to a new plate.
} Normalized gDNA (10 μl)
} TD (25 μl)
} TDE1 (15 μl)
4 Shake at 1800 rpm for 1 minute.
5 Centrifuge at 280 × g for 1 minute.
6 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.
Document # 1000000005003 v00
Clean Up Tagmented DNA
□1 Add 65 μl SPB.
□2 Shake at 1800 rpm for 1 minute.
□3 Incubate at room temperature for 8 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 on the magnetic stand for 10 minutes.
□10 Remove from the magnetic stand.
□11 Add 22.5 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 20 μl supernatant.
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.
TruSight Rapid Capture Checklist
Clean Up Amplified DNA
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer 50 μl supernatant.
□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 Add 27.5 μl RSB.
□13 Shake at 1800 rpm for 1 minute.
□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 25 μl supernatant.
□18 Quantify the library using a fluorometric method.
Hybridize Probes
□1
□2
□3
□4
□5
□6
Combine 500 ng of each DNA library. Make sure
that each library has a unique index.
} For total volume > 40 μl, concentrate the
pooled sample to 40 μl.
} For total volume < 40 μl, increase the volume
to 40 μl with RSB.
Add the following to a new plate.
} DNA library sample or pool (40 μl)
} EHB (50 μl)
} CSO (10 μl)
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.
Capture Hybridized Probes
□1 Centrifuge at 280 × g for 1 minute.
□2 Transfer all volumes.
□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 2 N NaOH, and then
vortex.
□12 Add 23.5 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.
□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 # 1000000005003 v00
For Research Use Only. Not for
use in diagnostic procedures.
TruSight Rapid Capture Checklist
Perform Second Hybridization
□1
□2
□3
□4
□5
Add the following.
} RSB (15 μl)
} EHB (50 μl)
} CSO (10 μl)
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 supernatant.
□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 2 N NaOH, and then
vortex.
□12 Add 23.5 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.
□18 Add 4 μl ET2.
□19 Shake at 1800 rpm for 1 minute.
□20 Centrifuge at 280 × g for 1 minute.
Document # 1000000005003 v00
January 2016
ILLUMINA PROPRIETARY
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 Add 27.5 μl RSB.
□11 Shake at 1800 rpm for 1 minute.
□12 Incubate at room temperature for 2 minutes.
□13 Centrifuge at 280 × g for 1 minute.
□14 Place on a magnetic stand until liquid is clear.
□15 Transfer 25 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 3 of 5
For Research Use Only. Not for
use in diagnostic procedures.
TruSight Rapid Capture Checklist
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 NEM
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.
□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 Add 32.5 μl RSB.
□13 Shake at 1800 rpm for 1 minute.
□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 30 μl supernatant.
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 # 1000000005003 v00
TruSight Rapid Capture Checklist
Acronyms
Acronym
Definition
Acronym
Definition
SMB
Streptavidin Magnetic Beads
EE1
Enrichment Elution Buffer 1
SPB
Sample Purification Beads
EHB
Enrichment Hybridization Buffer
ST
Stop Tagment Buffer
ET2
Elute Target Buffer 2
TD
Tagment DNA Buffer
EWS
Enrichment Wash Solution
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
NLT
Nextera Library Tagment Plate
PPC
PCR Primer Cocktail
RCO
Rapid Capture Oligos
RSB
Resuspension Buffer
Document # 1000000005003 v00
January 2016
ILLUMINA PROPRIETARY
For Research Use Only. Not for
use in diagnostic procedures.
Page 5 of 5
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement