TruSeq Amplicon - Cancer Panel Checklist (1000000006993 v01)

TruSeq Amplicon - Cancer Panel Checklist (1000000006993 v01)
TruSeq Amplicon - Cancer Panel Checklist
Hybridize Oligo Pool
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Add 5 µl ACD1 to 1 well of the HYP plate.
Add 5 µl gDNA to each remaining well.
Add 5 µl ACP1 to the well containing ACD1.
Add 5 µl AFP1 to each well containing gDNA.
Centrifuge at 1000 × g for 1 minute.
Add 40 µl OHS1. Pipette to mix.
Centrifuge at 1000 × g for 1 minute.
Place on the preheated heat block and incubate
for 1 minute.
Reset the temperature to 40°C and incubate for 80
minutes.
Document # 1000000006993 v01
Remove Unbound Oligos
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Make sure that the heat block has cooled to 40˚C.
Remove from the heat block.
Centrifuge at 1000 × g for 1 minute.
Transfer each sample to the FPU plate.
Cover and centrifuge at 2400 × g for 2 minutes.
Wash 2 times with 45 µl SW1.
Reassemble the FPU plate.
Add 45 µl UB1.
Cover and centrifuge at 2400 × g for 2 minutes.
February 2016
ILLUMINA PROPRIETARY
For Research Use Only. Not for
use in diagnostic procedures.
Extend and Ligate Bound Oligos
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Add 45 µl ELM3 to the FPU plate.
Incubate at 37°C for 45 minutes.
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TruSeq Amplicon - Cancer Panel Checklist
Amplify Libraries
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Arrange the Index 1 (i7) adapters in columns 1–
12.
2 Arrange the Index 2 (i5) adapters in rows A–H.
3 Place the plate on a TruSeq Index Plate Fixture.
4 Add 4 µl of each Index 1 (i7) adapter down each
column.
5 Add 4 µl of each Index 2 (i5) adapter across each
row.
6 Remove the FPU plate from the incubator and do
the following.
a Replace the aluminum foil seal with the
filter plate lid.
b Centrifuge at 2400 × g for 2 minutes.
c Add 25 µl 50 mM NaOH. Pipette to mix.
d Incubate at room temperature for 5 minutes.
7 Add 56 µl TDP1 to a full tube (2.8 ml) of PMM2.
Invert to mix.
8 Transfer 22 µl PMM2/TDP1 mixture to the IAP
plate.
9 Transfer eluted samples from the FPU plate to the
IAP plate.
10 Centrifuge at 1000 × g for 1 minute.
11 Transfer the IAP plate to the post-amplification
area.
12 Place on the preprogrammed thermal cycler and
run the PCR program.
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Clean Up Libraries
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Centrifuge the IAP plate at 1000 × g for 1 minute.
Run an aliquot of library and control on
4% agarose gel (5 µl) or Bioanalyzer (1 µl).
Add 45 µl AMPure XP beads to the CLP plate.
Transfer all the supernatant from the IAP plate to
the CLP plate.
Shake at 1800 rpm for 2 minutes.
Incubate at room temperature for 10 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Wash 2 times with 200 µl 80% EtOH.
Use a 20 µl pipette to remove residual EtOH.
Remove from the magnetic stand and air-dry for
10 minutes.
Add 30 µl EBT.
Shake at 1800 rpm for 2 minutes.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand until liquid is clear.
Transfer 20 µl supernatant from the CLP plate to
the LNP plate.
Centrifuge at 1000 × g for 1 minute.
For Research Use Only. Not for
use in diagnostic procedures.
Normalize Libraries
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For 96 samples, add 4.4 ml LNA1 to a new 15 ml
conical tube.
Use a P1000 pipette to resuspend LNB1.
Transfer 800 µl LNB1 to the tube of LNA1.
Add 45 µl LNA1/LNB1 to the LNP plate.
Shake at 1800 rpm for 30 minutes.
Place on a magnetic stand until liquid is clear.
Remove and discard all supernatant.
Remove from the magnetic stand.
Wash 2 times with 45 µl LNW1.
Use a 20 µl pipette to remove residual LNW1.
Remove from the magnetic stand.
Add 30 µl fresh 0.1 N NaOH.
Shake at 1800 rpm for 5 minutes.
Place the LNP plate on a magnetic stand until
liquid is clear.
Add 30 µl LNS1 to the SGP plate.
Transfer 30 µl supernatant from the LNP plate to
the SGP plate.
Centrifuge at 1000 × 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 30 days.
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.
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February 2016
ILLUMINA PROPRIETARY
Document # 1000000006993 v01
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq Amplicon - Cancer Panel Checklist
Pool Libraries
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Centrifuge at 1000 × g for 1 minute.
Transfer 5 µl of each library to an 8-tube strip.
Transfer the contents of the 8-tube strip to the
PAL tube. Pipette to mix.
Denature and dilute pooled libraries to the
loading concentration for the sequencing
instrument you are using. See the denature and
dilute libraries guide for your instrument.
Acronyms
Acronym
Definition
Definition
SGP
Storage Plate
ACD1
Amplicon Control DNA 1
SW1
Stringent Wash 1
ACP1
Amplicon Control Oligo Pool 1
TDP1
TruSeq DNA Polymerase 1
AFP1
Amplicon Fixed Panel 1
UB1
Universal Buffer 1
CLP
Clean-up Plate
EBT
Elution Buffer with Tris
ELM3
Extension Ligation Mix 3
FPU
Filter Plate Unit
HT1
Hybridization Buffer
HYP
Hybridization Plate
IAP
Indexed Amplification 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
OHS1
Oligo Hybridization for Sequencing
Reagent 1
PAL
PMM2
Document # 1000000006993 v01
Acronym
Pooled Amplicon Library
PCR Master Mix 2
February 2016
ILLUMINA PROPRIETARY
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