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TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
FOR RESEARCH USE ONLY
Date: __________________________
Illumina Kit Lot #: __________________________
Description: ______________________________________
_________________________________________________
NOTE
Unless familiar with the LS protocol in the latest version of the TruSeq Nano DNA Sample Preparation Guide
(part # 15041110), new or less experienced users are advised to follow the protocol in the guide before using this
Experienced User Card and Lab Tracking Form.
ILLUMINA PROPRIETARY
Catalog # FC-121-9011DOC
Part # 15041111 Rev. B
November 2013
Page 1 of 32
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Page 2 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
Item
Lot Number
A-Tailing Mix (ATL)
Lot #: _____________________
End Repair Mix 2 (ERP2)
Lot #: _____________________
Enhanced PCR Mix (EPM)
Lot #: _____________________
Ligation Mix 2 (LIG2)
Lot #: _____________________
PCR Primer Cocktail (PPC)
Lot #: _____________________
Resuspension Buffer (RSB)
Lot #: _____________________
Sample Purification Beads (SPB)
Lot #: _____________________
Stop Ligation Buffer (STL)
Lot #: _____________________
80% Ethanol
Date Prepared: ____________
Adapters
Lot Number
DNA Adapter Plate (DAP)
Lot #: _____________________
DNA Adapter Index 1 (AD001)
Lot #: _____________________
DNA Adapter Index 2 (AD002)
Lot #: _____________________
DNA Adapter Index 3 (AD003)
Lot #: _____________________
DNA Adapter Index 4 (AD004)
Lot #: _____________________
DNA Adapter Index 5 (AD005)
Lot #: _____________________
DNA Adapter Index 6 (AD006)
Lot #: _____________________
DNA Adapter Index 7 (AD007)
Lot #: _____________________
DNA Adapter Index 8 (AD008)
Lot #: _____________________
DNA Adapter Index 9 (AD009)
Lot #: _____________________
DNA Adapter Index 10 (AD010)
Lot #: _____________________
DNA Adapter Index 11 (AD011)
Lot #: _____________________
DNA Adapter Index 12 (AD012)
Lot #: _____________________
DNA Adapter Index 13 (AD013)
Lot #: _____________________
DNA Adapter Index 14 (AD014)
Lot #: _____________________
DNA Adapter Index 15 (AD015)
Lot #: _____________________
DNA Adapter Index 16 (AD016)
Lot #: _____________________
Part # 15041111 Rev. B
Page 3 of 32
Consumables
Consumables
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Consumables
Date/Time: _______________________________
Operator: _______________________________
Adapters
Lot Number
DNA Adapter Index 18 (AD018)
Lot #: _____________________
DNA Adapter Index 19 (AD019)
Lot #: _____________________
DNA Adapter Index 20 (AD020)
Lot #: _____________________
DNA Adapter Index 21 (AD021)
Lot #: _____________________
DNA Adapter Index 22 (AD022)
Lot #: _____________________
DNA Adapter Index 23 (AD023)
Lot #: _____________________
DNA Adapter Index 25 (AD025)
Lot #: _____________________
DNA Adapter Index 27 (AD027)
Lot #: _____________________
Page 4 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
If you are pooling, record information about your samples before beginning library preparation
for later use in data analysis.
If you are pooling using adapter index tubes, Illumina recommends arranging samples that
will be combined into a common pool in the same row. Include a common index in each
column.
If you are pooling with the DAP, arrange samples that will be pooled together in the same
orientation as the indices in the DAP.
Sample Sheet Name: ________________________________________________
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Part # 15041111 Rev. B
Page 5 of 32
Prepare Adapter Setup
Prepare Adapter Setup
TruSeq Nano DNA Sample Prep LS Protocol
Prepare Adapter Setup
Experienced User Card and Lab Tracking Form
Page 6 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
This process describes how to optimally fragment the gDNA depending on the downstream
application. Covaris shearing generates dsDNA fragments with 3' or 5' overhangs. The
fragmentation process is optimized to obtain final libraries with the following average insert
sizes:
Table 1 Insert Size Options
Insert Size
Input DNA Per Sample
Recommended Read Length
350 bp
550 bp
100 ng
≤ 2 x 101 bp
200 ng
≤ 2 x 151 bp*
* Read lengths greater than 2 x 151 bp produce a significantly higher percentage of overlapping read-pairs.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
-15°C to -25°C
(2°C to 8°C after
initial thaw)
Illumina
Sample Purification Beads (SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Barcode labels for:
• CFP (Covaris Fragmentation
Plate)
• CSP (Clean Up Sheared DNA
Plate)
• DNA (DNA Plate)
• IMP (Insert Modification Plate)
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plates
4
15°C to 30°C
User
Covaris tubes
1 per sample
15°C to 30°C
User
DNA samples
100 ng per sample for
a 350 bp insert size
or
200 ng per sample for
a 550 bp insert size
-15°C to -25°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
Make CFP
[_] 1
Illumina recommends quantifying gDNA samples using a fluorometric quantification
method that uses dsDNA binding dyes.
Part # 15041111 Rev. B
Page 7 of 32
Fragment DNA
Fragment DNA
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Fragment DNA
Date/Time: _______________________________
[_] 2
Operator: _______________________________
Normalize the gDNA samples with Resuspension Buffer to one of the following in each well
of the new 0.3 ml PCR plate labeled with the DNA barcode:
• 100 ng in a final volume of 52.5 µl for a 350 bp insert size
• 200 ng in a final volume of 52.5 µl for a 550 bp insert size
Fragment DNA
[_] 1
Shear one of the following amounts of gDNA sample by transferring 52.5 µl of each DNA
sample from the DNA plate to a separate, new Covaris tube:
• 100 ng for a 350 bp insert size
• 200 ng for a 550 bp insert size
Use the wells of the new 0.3 ml PCR plate labeled with CFP barcode or another device to
hold the Covaris tubes upright.
[_] 2
Centrifuge the CFP plate at 600 × g for 5 seconds.
[_] 3
Fragment the DNA using the following settings:
Table 2 Covaris S220 Settings
350 bp Insert
Setting
Duty factor
550 bp Insert
5%
Peak Incident Power
175 W
Cycles per burst
200
Duration
50 seconds
Mode
25 seconds
Frequency sweeping
Temperature
5.5° to 6°C
Table 3 Covaris M220 Settings
Setting
350 bp Insert
550 bp Insert
Duty factor
20%
Peak Incident Power
50 W
Cycles per burst
Duration
Temperature
200
65 seconds
45 seconds
20°C
Page 8 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
Setting
350 bp Insert
Duty cycle
550 bp Insert
10%
Intensity
5.0
2.0
Cycles per burst
200
Duration
45 seconds
Mode
Frequency sweeping
Displayed Power
S2—23 W
S2—9 W
E210—14 W
E210—7 W
Temperature
5.5° to 6°C
[_] 4
Centrifuge the CFP plate at 600 × g for 5 seconds.
[_] 5
Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the CSP barcode, using a single
channel pipette.
[_] 6
Proceed immediately to Clean Up Fragmented DNA.
Clean Up Fragmented DNA
[_] 1
Vortex the room temperature Sample Purification Beads for at least 1 minute or until they
are well dispersed.
[_] 2
Add 80 µl well-mixed Sample Purification Beads to each well of the CSP plate containing
50 µl of fragmented gDNA. Set a 200 µl pipette to 125 µl, and then gently pipette the entire
volume up and down 10 times to mix thoroughly.
[_] 3
Incubate the CSP plate at room temperature for 5 minutes.
Start time: _____________________
[_] 4
Stop time: _____________________
Place the CSP plate on the magnetic stand at room temperature for 8 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 5
Using a 200 µl single channel or multichannel pipette set to 125 µl, remove and discard
125 µl of the supernatant from each well of the CSP plate.
[_] 6
With the CSP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well without disturbing the beads.
[_] 7
Incubate the CSP plate at room temperature for 30 seconds, and then remove and discard all
of the supernatant from each well.
[_] 8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
[_] 9
With the CSP plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
Start time: _____________________
Part # 15041111 Rev. B
Stop time: _____________________
Page 9 of 32
Fragment DNA
Table 4 Covaris S2 and E210 Settings
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Fragment DNA
Date/Time: _______________________________
Operator: _______________________________
[_] 10 With the CSP plate on the magnetic stand, add 62.5 µl Resuspension Buffer to each well of
the plate.
[_] 11 Remove the CSP plate from the magnetic stand.
[_] 12 Resuspend the beads in each well of the CSP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently pipette
the entire volume up and down 10 times to mix thoroughly.
[_] 13 Incubate the CSP plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 14 Place the CSP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 15 Transfer 60 µl of the clear supernatant from each well of the CSP plate to the corresponding
well of the new 0.3 ml PCR plate labeled with the IMP barcode.
[_] 16 Proceed immediately to Perform End Repair and Size Selection on page 11.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 10 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
This process converts the overhangs resulting from fragmentation into blunt ends using End
Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the 5' to
3' polymerase activity fills in the 5' overhangs. Following end repair, the appropriate library size
is selected using different ratios of the Sample Purification Beads.
Consumables
Item
Quantity
Storage
Supplied By
End Repair Mix 2 (ERP2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Barcode labels for:
• ALP (Adapter Ligation Plate)
• CEP (Clean Up End Repair
Plate)
1 label per plate
15°C to 30°C
Illumina
15 ml conical tube
(when processing > 6 samples at a
time)
or
1.7 ml microcentrifuge tube
(when processing ≤ 6 samples at a
time)
1
15°C to 30°C
User
96-well 0.3 ml PCR plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Ice bucket
As needed
-15°C to -25°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
PCR grade water
1 bottle
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps (if using
multichannel pipettes)
6
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs (if using multichannel
pipettes)
6
15°C to 30°C
User
Make IMP
[_] 1
Centrifuge the thawed End Repair Mix 2 tube at 600 × g for 5 seconds.
Part # 15041111 Rev. B
Page 11 of 32
Perform End Repair and Size Selection
Perform End Repair and Size Selection
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Perform End Repair and Size Selection
Date/Time: _______________________________
Operator: _______________________________
[_] 2
Add 40 µl End Repair Mix 2 to each well of the IMP plate containing the fragmented DNA.
Set a 200 µl pipette to 95 µl, and then gently pipette the entire volume up and down
10 times to mix thoroughly.
[_] 3
Seal the IMP plate with a Microseal ‘B’ adhesive seal.
[_] 4
Return the End Repair Mix 2 tube to -15°C to -25°C storage.
Incubate IMP
[_] 1
Place the sealed IMP plate on the pre-programmed thermal cycler. Close the lid then
select and run the ERP program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 30°C for 30 minutes
[_] c Hold at 4°C
[_] 2
Remove the IMP plate from the thermal cycler when the program reaches 4°C.
Clean Up IMP and Size Selection
[_] 1
Remove the adhesive seal from the IMP plate.
Remove Large DNA Fragments
[_] 1
Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed.
[_] 2
Add the Sample Purification Beads and PCR grade water to one of the following tubes, to
create a diluted bead mixture of 160 µl per 100 µl of end-repaired sample:
• New 15 ml conical tube, when processing > 6 samples at a time
• New 1.7 ml microcentrifuge tube, when processing ≤ 6 samples at a time
Determine the volumes using the following formulas, which include 15% excess for multiple
samples:
Table 5 Diluted Bead Mixture for a 350 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
109.25 µl
# of samples X
74.75 µl
Table 6 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
92 µl
# of samples X
92 µl
Page 12 of 32
Example
Amount
per 12 samples
Your Calculation
1311 µl
897 µl
Example
Amount
per 12 samples
1104 µl
Your Calculation
1104 µl
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
Vortex the diluted bead mixture for 5 seconds to make sure that the beads are evenly
dispersed.
[_] 4
Add 160 µl of the diluted bead mixture to each well of the IMP plate containing 100 µl of
the end repaired sample. Set a 200 µl pipette to 200 µl, and then gently pipette the entire
volume up and down 10 times to mix thoroughly.
[_] 5
Incubate the IMP plate at room temperature for 5 minutes.
Start time: _____________________
[_] 6
Stop time: _____________________
Place the IMP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 7
Use a 200 µl single channel or multichannel pipette set to 125 µl to transfer 125 µl of the
supernatant, containing the DNA of interest, from each well of the IMP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the CEP barcode.
[_] 8
Repeat step 7 one time.
[_] 9
Discard the IMP plate containing the beads.
[_] 10 Discard any remaining diluted bead mixture.
Remove Small DNA Fragments
[_] 1
Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed.
[_] 2
Add 30 µl undiluted Sample Purification Beads to each well of the CEP plate containing
250 µl of the supernatant with the DNA of interest. Set a 200 µl pipette to 200 µl, and then
gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Incubate the CEP plate at room temperature for 5 minutes.
Start time: _____________________
[_] 4
Stop time: _____________________
Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 5
Using a 200 µl single channel or multichannel pipette set to 138 µl, remove and discard
138 µl of the supernatant from each well of the CEP plate.
[_] 6
Repeat step 5 one time, removing and discarding a total of 276 µl of the supernatant from
each well.
[_] 7
With the CEP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well without disturbing the beads.
[_] 8
Incubate the CEP plate at room temperature for 30 seconds, and then remove and discard all
of the supernatant from each well.
[_] 9
Repeat steps 7 and 8 one time for a total of two 80% EtOH washes.
[_] 10 With the CEP plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
Start time: _____________________
Part # 15041111 Rev. B
Stop time: _____________________
Page 13 of 32
Perform End Repair and Size Selection
[_] 3
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Perform End Repair and Size Selection
Date/Time: _______________________________
Operator: _______________________________
[_] 11 With the CEP plate on the magnetic stand, add 20 µl Resuspension Buffer to each well of the
plate.
[_] 12 Remove the CEP plate from the magnetic stand.
[_] 13 Resuspend the beads in each well of the CEP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently pipette
the entire volume up and down 10 times to mix thoroughly.
[_] 14 Incubate the CEP plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 15 Place the CEP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 16 Transfer 17.5 µl of the clear supernatant from each well of the CEP plate to the
corresponding well of the new 0.3 ml PCR plate labeled with the ALP barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Adenylate 3' Ends on page 15, the protocol can be
safely stopped here. If you are stopping, seal the ALP plate with a Microseal ‘B’ adhesive seal and
store at -15°C to -25°C for up to 7 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 14 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to one another during the adapter ligation reaction. A corresponding single
‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the
adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template)
formation.
Consumables
Item
Quantity
Storage
Supplied By
A-Tailing Mix (ATL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps
(if using multichannel pipettes)
2
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
2
15°C to 30°C
User
Add ATL
[_] 1
Centrifuge the thawed A-Tailing Mix tube at 600 × g for 5 seconds.
[_] 2
Add 12.5 µl thawed A-Tailing Mix to each well of the ALP plate. Set a 20 µl pipette to 20 µl,
then gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
[_] 4
Return the A-Tailing Mix tube to -15° to -25°C storage.
Incubate 1 ALP
[_] 1
Place the sealed ALP plate, containing 30 µl of each sample, on the pre-programmed
thermal cycler. Close the lid, then select and run the ATAIL70 program.
[_] a Choose the pre-heat lid option and set to 100°C
[_] b 37°C for 30 minutes
[_] c 70°C for 5 minutes
[_] d 4°C for 5 minutes
[_] e Hold at 4°C
[_] 2
When the thermal cycler temperature has been at 4°C for 5 minutes, remove the ALP plate
from the thermal cycler.
Start time: _____________________
Part # 15041111 Rev. B
Stop time: _____________________
Page 15 of 32
Adenylate 3' Ends
Adenylate 3' Ends
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Adenylate 3' Ends
Date/Time: _______________________________
Operator: _______________________________
[_] 3
Centrifuge the ALP plate at 280 × g for 1 minute.
[_] 4
Proceed immediately to Ligate Adapters on page 17.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 16 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
This process ligates multiple indexing adapters to the ends of the DNA fragments, preparing
them for hybridization onto a flow cell.
Consumables
Item
Quantity
Storage
Supplied By
Choose from the following
depending on the kit you are
using:
• TruSeq Nano DNA LT Sample
Prep Kit contents:
• DNA Adapter Indices
(AD001–AD016, AD018–
AD023, AD025, AD027)
• TruSeq Nano DNA HT Sample
Prep Kit contents:
• DAP (DNA Adapter Plate)
1 tube of each index
being used, per
column of 8 reactions
or
1 DAP
-15°C to -25°C
Illumina
Ligation Mix 2 (LIG2)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Stop Ligation Buffer (STL)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Barcode labels for:
• CAP (Clean Up ALP Plate)
• DAP (DNA Adapter Plate)
(if using the HT kit)
• PCR (Polymerase Chain
Reaction Plate)
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
800 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps (if using
multichannel pipettes)
3–27
15°C to 30°C
User
Part # 15041111 Rev. B
Page 17 of 32
Ligate Adapters
Ligate Adapters
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Ligate Adapters
Date/Time: _______________________________
Operator: _______________________________
Item
Quantity
Storage
Supplied By
RNase/DNase-free reagent
reservoirs (if using multichannel
pipettes)
3–27
15°C to 30°C
User
Add LIG
[_] 1
Do one of the following:
• If using DNA Adapter tubes, centrifuge the thawed tubes at 600 × g for 5 seconds.
• If using a DAP:
— Thaw the plate for 10 minutes at room temperature on the benchtop. Visually
inspect the wells to make sure that they all are thawed.
Start time: _____________________
Stop time: _____________________
— Remove the adapter plate tape seal.
— Centrifuge the plate at 280 × g for 1 minute to collect all of the adapter to the bottom
of the well.
— Remove the plastic cover. Save the cover if you are not processing the entire plate at
one time.
— If it is the first time using this DAP, apply the DAP barcode label to the plate.
NOTE
• The DAP is single-use for each well.
• Illumina recommends that the DAP does not undergo more than 4 freeze-thaw cycles.
[_] 2
Centrifuge the Stop Ligation Buffer tube at 600 × g for 5 seconds.
[_] 3
Immediately before use, remove the Ligation Mix 2 tube from -15°C to -25°C storage.
[_] 4
Remove the adhesive seal from the ALP plate.
[_] 5
Add 2.5 µl Resuspension Buffer to each well of the ALP plate.
[_] 6
Add 2.5 µl Ligation Mix 2 to each well of the ALP plate.
[_] 7
Return the Ligation Mix 2 tube to -15°C to -25°C storage immediately after use.
[_] 8
Do one of the following:
• If using DNA Adapter tubes, add 2.5 µl thawed DNA Adapter Index to each well of the
ALP plate. Set a 200 µl pipette to 35 µl, then gently pipette the entire volume up and
down 10 times to mix thoroughly.
• If using a DAP:
— Place the DAP on the benchtop so that the part number barcode, on the long side of
the plate, is facing you and the clipped corner is on the lower left.
— Do one of the following to pierce the foil seal:
— If using the entire plate at one time, use the bottom of a clean 96-well semiskirted PCR plate to pierce a hole in all of the well seals simultaneously. Gently,
but firmly, press the clean plate over the foil seal.
— If using only part of the plate, use the bottom of a clean eight-tube strip, with
caps attached, to pierce holes in the seals of the wells that will be used for
ligation. Repeat with a new, clean eight-tube strip, with caps attached, for each
row or column of adapters that will be used for ligation.
Page 18 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
[_] 9
Seal the ALP plate with a Microseal ‘B’ adhesive seal.
[_] 10 Centrifuge the ALP plate at 280 × g for 1 minute.
Incubate 2 ALP
[_] 1
Place the sealed ALP plate, containing 37.5 µl of each sample, on the pre-programmed
thermal cycler. Close the lid then select and run the LIG program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 30°C for 10 minutes
[_] c Hold at 4°C
[_] 2
Remove the ALP plate from the thermal cycler when the program reaches 4°C.
Add STL
[_] 1
Remove the adhesive seal from the ALP plate.
[_] 2
Add 5 µl Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation. Set a
200 µl pipette to 40 µl, then gently pipette the entire volume up and down 10 times to mix
thoroughly.
Clean Up ALP
[_] 1
Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed.
[_] 2
Add 42.5 µl well-mixed Sample Purification Beads to each well of the ALP plate. Set a
200 µl pipette to 75 µl, and then gently pipette the entire volume up and down 10 times to
mix thoroughly.
[_] 3
Incubate the ALP plate at room temperature for 5 minutes.
Start time: _____________________
[_] 4
Stop time: _____________________
Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 5
Remove and discard 80 µl of the supernatant from each well of the ALP plate.
[_] 6
With the ALP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well without disturbing the beads.
[_] 7
Incubate the ALP plate at room temperature for 30 seconds, and then remove and discard all
of the supernatant from each well.
[_] 8
Repeat steps 6 and 7 one time for a total of two 80% EtOH washes.
[_] 9
With the ALP plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
Start time: _____________________
Part # 15041111 Rev. B
Stop time: _____________________
Page 19 of 32
Ligate Adapters
— Using an eight-tip multichannel pipette, transfer 2.5 µl thawed DNA Adapter from
the DAP well to each well of the ALP plate. Set a 200 µl pipette to 35 µl, then gently
pipette the entire volume up and down 10 times to mix thoroughly.
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Ligate Adapters
Date/Time: _______________________________
Operator: _______________________________
[_] 10 With the ALP plate on the magnetic stand, add 52.5 µl Resuspension Buffer to each well of
the plate.
[_] 11 Remove the ALP plate from the magnetic stand.
[_] 12 Resuspend the beads in each well of the ALP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently pipette
the entire volume up and down 10 times to mix thoroughly.
[_] 13 Incubate the ALP plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 14 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 15 Transfer 50 µl of the clear supernatant from each well of the ALP plate to the corresponding
well of the new 0.3 ml PCR plate labeled with the CAP barcode.
[_] 16 Vortex the Sample Purification Beads until they are well dispersed.
[_] 17 Add 50 µl mixed Sample Purification Beads to each well of the CAP plate for a second
cleanup. Set a 200 µl pipette to 90 µl, and then gently pipette the entire volume up and
down 10 times to mix thoroughly.
[_] 18 Incubate the CAP plate at room temperature for 5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 19 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 20 Remove and discard 95 µl of the supernatant from each well of the CAP plate.
[_] 21 With the CAP plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well.
[_] 22 Incubate the CAP plate at room temperature for 30 seconds, and then remove and discard all
of the supernatant from each well.
[_] 23 Repeat steps 21 and 22 one time for a total of two 80% EtOH washes.
[_] 24 With the CAP plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Remove and discard any remaining EtOH with a 10 µl pipette.
Start time: _____________________
Stop time: _____________________
[_] 25 With the CAP plate on the magnetic stand, add 27.5 µl Resuspension Buffer to each well of
the plate.
[_] 26 Remove the CAP plate from the magnetic stand.
[_] 27 Resuspend the beads in each well of the CAP plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently pipette
the entire volume up and down 10 times to mix thoroughly.
[_] 28 Incubate the CAP plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
Page 20 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
Start time: _____________________
Stop time: _____________________
[_] 30 Transfer 25 µl of the clear supernatant from each well of the CAP plate to the corresponding
well of the new 0.3 ml PCR plate labeled with the PCR barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Enrich DNA Fragments on page 23, you can safely
stop the protocol here. If you are stopping, seal the PCR plate with a Microseal ‘B’ adhesive seal
and store at -15°C to -25°C for up to 7 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Part # 15041111 Rev. B
Page 21 of 32
Ligate Adapters
[_] 29 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
TruSeq Nano DNA Sample Prep LS Protocol
Ligate Adapters
Experienced User Card and Lab Tracking Form
Page 22 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
This process uses PCR to selectively enrich those DNA fragments that have adapter molecules
on both ends and to amplify the amount of DNA in the library. The PCR is performed with a
PCR Primer Cocktail that anneals to the ends of the adapters. Minimize the number of PCR
cycles to avoid skewing the representation of the library.
Consumables
Item
Quantity
Storage
Supplied By
Enhanced PCR Mix (EPM)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
PCR Primer Cocktail (PPC)
LT kit - 1 tube per 24
reactions
or
HT kit - 1 tube per 48
reactions
-15°C to -25°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
TSP1 (Target Sample Plate)
barcode label
1 label per plate
15°C to 30°C
Illumina
96-well 0.3 ml PCR plate
1
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
RNase/DNase-free eight-tube
strips and caps
(if using multichannel pipettes)
5
15°C to 30°C
User
RNase/DNase-free reagent
reservoirs
(if using multichannel pipettes)
5
15°C to 30°C
User
Make PCR
The following procedure assumes the following amount of input DNA sample for library
preparation and is designed to result in high library yields:
} 100 ng for a 350 bp insert size
} 200 ng for a 550 bp insert size
[_] 1
Add 5 µl thawed PCR Primer Cocktail to each well of the PCR plate.
[_] 2
Add 20 µl thawed Enhanced PCR Mix to each well of the PCR plate. Set a 200 µl pipette to
40 µl, then gently pipette the entire volume up and down 10 times to mix thoroughly.
Part # 15041111 Rev. B
Page 23 of 32
Enrich DNA Fragments
Enrich DNA Fragments
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Enrich DNA Fragments
Date/Time: _______________________________
[_] 3
Operator: _______________________________
Seal the PCR plate with a Microseal ‘B’ adhesive seal.
Amp PCR
[_] 1
Place the sealed PCR plate, containing 50 µl of each sample, on the pre-programmed
thermal cycler. Close the lid, then select and run PCRNano to amplify the plate.
[_] a Choose the pre-heat lid option and set to 100°C
[_] b 95°C for 3 minutes
[_] c 8 cycles of:
— 98°C for 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
[_] d 72°C for 5 minutes
[_] e Hold at 4°C
Clean Up PCR
[_] 1
Centrifuge the PCR plate at 280 × g for 1 minute.
[_] 2
Remove the adhesive seal from the PCR plate.
[_] 3
Vortex the Sample Purification Beads until they are well dispersed.
[_] 4
Do one of the following, depending on the adapter type used:
• If using the DNA Adapter tubes, add 50 µl mixed Sample Purification Beads to each
well of the PCR plate containing 50 µl of the PCR amplified library. Set a 200 µl pipette
to 90 µl, then gently pipette the entire volume up and down 10 times to mix thoroughly.
• If using the DAP, add 47.5 µl mixed Sample Purification Beads to each well of the PCR
plate containing 50 µl of the PCR amplified library. Set a 200 µl pipette to 90 µl, then
gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 5
Incubate the PCR plate at room temperature for 5 minutes.
Start time: _____________________
[_] 6
Stop time: _____________________
Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 7
Remove and discard 95 µl of the supernatant from each well of the PCR plate.
[_] 8
With the PCR plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each
well without disturbing the beads.
[_] 9
Incubate the PCR plate at room temperature for 30 seconds, and then remove and discard all
of the supernatant from each well.
[_] 10 Repeat steps 8 and 9 one time for a total of two 80% EtOH washes.
[_] 11 With the PCR plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes. Remove and discard any remaining EtOH from each well of the PCR plate with a
10 µl pipette.
Start time: _____________________
Stop time: _____________________
Page 24 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
[_] 13 Remove the PCR plate from the magnetic stand.
[_] 14 Resuspend the beads in each well of the PCR plate by repeatedly dispensing the
Resuspension Buffer over the bead pellet until it is immersed in the solution. Gently pipette
the entire volume up and down 10 times to mix thoroughly.
[_] 15 Incubate the PCR plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 16 Place the PCR plate on the magnetic stand at room temperature for 5 minutes or until the
liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 17 Transfer 30 µl of the clear supernatant from each well of the PCR plate to the corresponding
well of the new 0.3 ml PCR plate labeled with the TSP1 barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Library on page 27, you can safely stop the
protocol here. If you are stopping, seal the TSP1 plate with a Microseal ‘B’ adhesive seal and store
at -15°C to -25°C for up to 7 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Part # 15041111 Rev. B
Page 25 of 32
Enrich DNA Fragments
[_] 12 With the PCR plate on the magnetic stand, add 32.5 µl Resuspension Buffer to each well of
the PCR plate.
TruSeq Nano DNA Sample Prep LS Protocol
Enrich DNA Fragments
Experienced User Card and Lab Tracking Form
Page 26 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
Illumina recommends performing the following procedures for quality control analysis on your
sample library and quantification of the DNA library templates.
Quantify Libraries
Quantify your libraries using a fluorometric quantification method that uses dsDNA binding
dyes or qPCR.
[Optional] Quality Control
To verify the size of your fragments, check the template size distribution. Run samples on a
Bioanalyzer for qualitative purposes only.
Do one of the following:
• If using a High Sensitivity DNA chip:
— Prepare a 1:100 dilution of the DNA library with water.
— Run 1 µl of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzer.
• If using a DNA 7500 chip, run 1 µl of undiluted DNA library on an Agilent
Technologies 2100 Bioanalyzer.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Part # 15041111 Rev. B
Page 27 of 32
Validate Library
Validate Library
TruSeq Nano DNA Sample Prep LS Protocol
Validate Library
Experienced User Card and Lab Tracking Form
Page 28 of 32
Part # 15041111 Rev. B
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Date/Time: _______________________________
Operator: _______________________________
This process describes how to prepare DNA templates for cluster generation. Indexed DNA
libraries are normalized to 10 nM in the DCT plate and then pooled in equal volumes in the
PDP plate. DNA libraries not intended for pooling are normalized to 10 nM in the DCT plate.
Consumables
Item
Quantity
Storage
Supplied By
Barcode labels for:
• DCT (Diluted Cluster Template)
• PDP (Pooled DCT Plate)
(for pooling only)
1 label per plate
15°C to 30°C
Illumina
1.7 ml microcentrifuge tube
(when processing > 48 samples at
a time)
1
15°C to 30°C
User
96-well MIDI plates
2
(second plate for
pooling only, if
pooling > 40 samples)
15°C to 30°C
User
96-well 0.3 ml PCR plate
(for pooling only, if pooling ≤ 40
samples)
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Tris-HCl 10 mM, pH8.5
with 0.1% Tween 20
Enough to normalize
the concentration of
each sample library to
10 nM
15°C to 30°C
User
Make DCT
[_] 1
Transfer 10 µl of sample library from each well of the TSP1 plate to the corresponding well
of the new MIDI plate labeled with the DCT barcode.
[_] 2
Normalize the concentration of sample library in each well of the DCT plate to 10 nM using
Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
[_] 3
Gently pipette the entire normalized sample library volume up and down 10 times to mix
thoroughly.
[_] 4
Depending on the type of library you want to generate, do one of the following:
• For non-pooled libraries, the protocol stops here. Do one of the following:
— Proceed to cluster generation.
— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at -15°C to -25°C.
• For pooled libraries, proceed to Make PDP (for pooling only).
Part # 15041111 Rev. B
Page 29 of 32
Normalize and Pool Libraries
Normalize and Pool Libraries
TruSeq Nano DNA Sample Prep LS Protocol
Experienced User Card and Lab Tracking Form
Normalize and Pool Libraries
Date/Time: _______________________________
Operator: _______________________________
Make PDP (for pooling only)
[_] 1
Determine the number of samples to be combined together for each pool.
[_] 2
Do one of the following:
• If pooling 2–24 samples:
— Transfer 10 µl of each normalized sample library to be pooled from the DCT plate to
one well of the new 0.3 ml PCR plate labeled with the PDP barcode.
The total volume in each well of the PDP plate is 10 X the number of combined sample
libraries and 20–240 µl (2–24 libraries).
• If pooling 25–48 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library in
column 1 of the DCT plate to column 1 of the new 0.3 ml PCR or MIDI plate labeled
with the PDP barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate to
column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the DCT
plate. The result is a PDP plate with pooled samples in column 1. Gently pipette the
entire volume of each well of column 1 up and down 10 times to mix thoroughly.
— Combine the contents of each well of column 1 into well A2 of the PDP plate for the
final pool.
• If pooling 49–96 samples:
— Using a multichannel pipette, transfer 5 µl of each normalized sample library in
column 1 of the DCT plate to column 1 of the new MIDI plate labeled with the PDP
barcode.
— Transfer 5 µl of each normalized sample library in column 2 of the DCT plate to
column 1 of the PDP plate.
— Repeat the transfer for as many times as there are remaining columns in the DCT
plate. The result is a PDP plate with pooled samples in column 1. Gently pipette the
entire volume of each well of column 1 up and down 10 times to mix thoroughly.
— Combine the contents of each well of column 1 into a 1.7 ml microcentrifuge tube for
the final pool.
[_] 3
Gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 4
Do one of the following:
• Proceed to cluster generation.
• Do one of the following, depending on the item that contains the final pool:
— Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -15°C to -25°C.
— Cap the 1.7 ml microcentrifuge tube and store at -15°C to -25°C.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 30 of 32
Part # 15041111 Rev. B
Technical Assistance
For technical assistance, contact Illumina Technical Support.
Table 7 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 8 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at www.illumina.com/msds.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go to
www.illumina.com/support, select a product, then click Documentation & Literature.
Part # 15041111 Rev. B
Page 31 of 32
*15041111*
Part # 15041111 Rev. B
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com