TruSeq DNA PCR-Free Library Prep Checklist (15075700 A)

TruSeq DNA PCR-Free Library Prep Checklist (15075700 A)
TruSeq DNA PCR-Free Library Prep
Fragment DNA
□1
□2
□3
□4
□5
□6
Normalize gDNA with RSB to 55 µl in the DNA
plate.
• 20 ng/µl for a 350 bp insert size
• 40 ng/µl for a 550 bp insert size
Mix thoroughly.
Centrifuge.
Transfer 52.5 µl DNA to Covaris tubes.
Centrifuge at 280 × g for 5 seconds.
Fragment the DNA using the following settings.
□12 Place on a magnetic stand until liquid is clear.
□13 Remove and discard all supernatant.
□14 Wash 2 times with 200 µl 80% EtOH.
□15 Use a 20 µl pipette to remove residual EtOH.
□16 Air-dry for 5 minutes.
□17 Add 52.5 µl RSB.
□18 Remove from the magnetic stand and mix.
□19 Incubate at room temperature for 2 minutes.
□20 [HS] Centrifuge at 280 × g for 1 minute.
□21 Place on a magnetic stand until liquid is clear.
□22 Transfer 50 µl supernatant to the IMP plate.
Table 2 550 bp Insert Settings
M220 S220
S2
E210
Setting
Duty Factor (%)
20
5
10
Intensity
—
—
2.0
Power (W)
50
175
9
7
Cycles/Burst
200
Duration (sec)
45
25
45
Mode
—
Frequency sweeping
Temperature (°C)
20
5.5–6
□7 Centrifuge at 280 × g for 5 seconds.
□8 Transfer 50 µl supernatant to the CSP plate.
□9 Add 80 µl SPB and mix.
□10 Incubate at room temperature for 5 minutes.
□11 [HS] Centrifuge at 280 × g for 1 minute.
Part # 15075700 Rev. A
Repair Ends and Select Library Size
□1
□2
□3
□4
□5
□6
□7
□8
□9
□10
□11
□12
□13
□14
□15
□16
□17
□18
□19
□20
□21
□22
□23
□24
□25
Table 1 350 bp Insert Settings
M220 S220
S2
E210
Setting
Duty Factor (%)
20
5
10
Intensity
—
—
5.0
Power (W)
50
175
23
14
Cycles/Burst
200
Duration (sec)
65
50
45
Mode
—
Frequency sweeping
Temperature (°C)
20
5.5–6
June 2015
ILLUMINA PROPRIETARY
For Research Use Only. Not for
use in diagnostic procedures.
Add 10 µl CTE.
Add 40 µl ERP2/ERP3 and mix.
[HS] Centrifuge at 280 × g for 1 minute.
Incubate as follows.
• [HS] Place on the 30°C microheating system for
30 minutes, and then place on ice.
• [LS] Place on the thermal cycler and run the
ERP program.
Dilute SPB with PCR grade water to 160 µl per
100 µl of sample.
Vortex diluted SPB until well-dispersed.
Add 160 µl diluted SPB and mix.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Transfer 250 µl supernatant to the CEP plate.
Add 30 µl SPB and mix.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
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.
Air-dry for 5 minutes.
Add 17.5 µl RSB.
Remove from the magnetic stand and mix.
Incubate at room temperature for 2 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand until liquid is clear.
Transfer 15 µl supernatant to the ALP plate.
Page 1 of 3
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq DNA PCR-Free Library Prep
Adenylate 3ʹ Ends
□1
□2
□3
Add 2.5 µl CTA.
Add 12.5 µl ATL/ATL2 and mix.
Incubate as follows.
[HS]
a Place on the 37°C microheating system for
30 minutes.
b Move to the 70°C microheating system for
5 minutes.
c Place on ice for 5 minutes.
[LS]
a Place on the thermal cycler and run the
ATAIL70 program.
□1
Add the following and mix.
Reagent
CTL
LIG2
DNA adapters
□
□
□
□7
□8
Ligate Adapters
□2
□3
□
□4
□5
□6
Volume (µl)
2.5
2.5
2.5
□9
Transfer 50 µl supernatant to the CAP plate.
Repeat steps 6a through 6m with the new plate
using the Round 2 volumes.
Transfer 20 µl supernatant to the TSP1 plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 7 days.
Centrifuge at 280 × g for 1 minute.
Incubate as follows.
• [HS] Place on the 30°C microheating system for
10 minutes, and then place on ice.
• [LS] Place on the thermal cycler and run the
LIG program.
Add 5 µl STL and mix.
[HS] Centrifuge at 280 × g for 1 minute.
Perform steps 6a through 6m using the Round 1
volumes.
a Add SPB and mix.
□
SPB
Round 1
42.5 µl
Round 2
50 µl
□b Incubate at room temperature for 5 minutes.
□c [HS] Centrifuge at 280 × g for 1 minute.
□d Place on a magnetic stand until liquid is clear.
□e Remove and discard all supernatant.
□f Wash 2 times with 200 µl 80% EtOH.
□g Use a 20 µl pipette to remove residual EtOH.
□h Air-dry for 5 minutes.
□i Add RSB.
RSB
Round 1
52.5 µl
Round 2
22.5 µl
□j Remove from the magnetic stand and mix.
□k Incubate at room temperature for 2 minutes.
□l [HS] Centrifuge at 280 × g for 1 minute.
□mPlace on a magnetic stand until liquid is clear.
Page 2 of 3
June 2015
ILLUMINA PROPRIETARY
Part # 15075700 Rev. A
For Research Use Only. Not for
use in diagnostic procedures.
TruSeq DNA PCR-Free Library Prep
Validate Libraries
□1
□
Quantify the libraries using qPCR, with the
following modifications.
} Use at least 2 µl of the original library stock.
} Perform 2 additional dilutions.
} Determine the concentration of the diluted library.
} Perform a size adjustment calculation.
} Calculate the concentration of the undiluted
library.
2 Verify fragment size by checking the library size
distribution.
a Dilute the DNA library 1:5 with water.
b Run 1 µl diluted DNA library on a High
Sensitivity DNA chip.
□
□
Normalize and Pool Libraries
□1
□2
□3
□4
□5
□6
□7
□8
Transfer 5 µl library to the DCT plate.
Normalize with Tris-HCl 10 mM, pH 8.5 with
0.1% Tween 20 to 2 nM and mix.
[HS] Centrifuge at 280 × g for 1 minute.
If pooling 2–24 samples, transfer 5 µl to a single
well of the PDP plate.
If pooling 25–96 samples.
a Transfer 5 µl to column 1 of the PDP plate and
mix.
b [HS] Centrifuge at 280 × g for 1 minute.
c Transfer column 1 to well A2.
Mix thoroughly.
[HS] Centrifuge at 280 × g for 1 minute.
Proceed to cluster generation.
Acronym
Definition
ALP
Adapter Ligation Plate
ATL
A-Tailing Mix
CAP
Clean Up ALP Plate
□
CEP
Clean Up End Repair Plate
□
□
CSP
Clean Up Sheared DNA Plate
CTA
A-Tailing Control
CTE
End Repair Control
CTL
Ligation Control
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template Plate
DNA
Customer Sample DNA Plate
ERP
End Repair Mix
IMP
Insert Modification Plate
LIG
Ligation Mix
PDP
Pooled Dilution Plate
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
SAFE STOPPING POINT
If you are stopping, seal the plate and store at
-25°C to -15°C.
Part # 15075700 Rev. A
Acronyms
June 2015
ILLUMINA PROPRIETARY
Page 3 of 3
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