iDeal Library Preparation kit(incl. Index Primer Set 1)
Cat. No. C05010020 (24 rxns)

USER GUIDE
Version 2 03_2019

The iDeal Library Preparation Kit has been validated
on IP-Star Compact Automated System. Two versionsof protocol (manual and automated) are described inthis manual.

Please read this manual carefully
before starting your experimentContents
Kit materials

4

Required materials not provided

6

Manual processing

7

Automated processing

16

Related products

28

3

Kit materials
The iDeal Library Preparation Kit contains all reagents necessary for the
preparation of 24 libraries. The kit contains also the indexes allowing
for multiplexing up to 12 samples. The set of additional 12 indexes isavailable separately (Index Primer Set 2 (iDeal Library Preparation Kitx24), Diagenode, Cat. No. C05010021) allowing for multiplexing up to 24
samples.
Table 1. Reagents supplied with the iDeal Library Preparation Kit.
Description

4

StorageiDeal Library End Repair/dA-Tailing Enzyme Mix (green)-20°C

iDeal Library End Repair/dA-Tailing Buffer (green)-20°C

iDeal Library Ligation Master Mix (red)-20°C

iDeal Library Ligation Enhancer (red)-20°C

iDeal Library PCR Master Mix (blue)-20°C

iDeal Library Adaptor for Illumina (red)-20°C

iDeal Library Uracil Excision Reagent (red)-20°C

iDeal Library Universal PCR Primer for Illumina (blue)-20°C

IDEAL LIBRARY PREPARATION KIT MANUALTable 2. List of indexes supplied with the iDeal Library Preparation Kit.
Product

Index Primer Sequence

Expected
index Primer
Sequence Read

Quantity

StorageiDeal Library Index 1

5´-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATCACG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CGATGT

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TTAGGC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TGACCA

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ACAGTG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GCCAAT

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CAGATC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ACTTGA

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GATCAG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TAGCTT

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GGCTAC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CTTGTA

10 µl

-20°C

Primer for Illumina
iDeal Library Index 2
Primer for Illumina
iDeal Library Index 3
Primer for Illumina
iDeal Library Index 4
Primer for Illumina
iDeal Library Index 5
Primer for Illumina
iDeal Library Index 6
Primer for Illumina
iDeal Library Index 7
Primer for Illumina
iDeal Library Index 8
Primer for Illumina
iDeal Library Index 9
Primer for Illumina
iDeal Library Index 10
Primer for Illumina
iDeal Library Index 11
Primer for Illumina
iDeal Library Index 12
Primer for Illumina

Where -s- indicates phosphorothioate bond.
NOTE: If fewer than 12 indexes are used in a lane for sequencing, it isrecommended to use the following indexes:-- Pool of 2 samples: Index #6 and 12-- Pool of 3 samples: Index #4, 6 and 12-- Pool of 6 samples: Index #2, 4, 5, 6, 7 and 12

5

Required materials not provided• 100% Ethanol• Nuclease-free Water• 10 mM Tris-HCl, pH 8.0 or 0.1X TE• DNA LoBind Tubes (Eppendorf #022431021)• AMPure® XP Beads (Beckman Coulter, Inc. #A63881)• Magnetic rack -DiaMag 0.2 ml (Diagenode, Cat. No. B04000001)• PCR Machine• Optional: Index Primer Set 2 (iDeal Library Preparation Kit x24)
(Diagenode, Cat. No. C05010021)
Table 3. List of indexes included in the kit Index Primer Set 2 (iDeal Library
Preparation Kit x24).
Product

Index Primer Sequence

Expected
index Primer
Sequence Read

Quantity

StorageiDeal Library Index 13

5´-CAAGCAGAAGACGGCATACGAGATTGTTGACTGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

AGTCAA

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATACGGAACTGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

AGTTCC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTCTGACATGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATGTCA

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATCGGGACGGGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CCGTCC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATGTGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GTCCGC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATCGTTTCACGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3

GTGAAA

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GTGGCC

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTCCGAAACGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GTTTCG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATTACGTACGGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CGTACG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATATCCACTCGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GAGTGG

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ACTGAT

10 µl

-20°C

5´-CAAGCAGAAGACGGCATACGAGATAAAGGAATGTG
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATTCCT

10 µl

-20°C

Primer for Illumina
iDeal Library Index 14
Primer for Illumina
iDeal Library Index 15
Primer for Illumina
iDeal Library Index 16
Primer for Illumina
iDeal Library Index 18
Primer for Illumina
iDeal Library Index 19
Primer for Illumina
iDeal Library Index 20
Primer for Illumina
iDeal Library Index 21
Primer for Illumina
iDeal Library Index 22
Primer for Illumina
iDeal Library Index 23
Primer for Illumina
iDeal Library Index 25
Primer for Illumina
iDeal Library Index 27
Primer for Illumina

6

IDEAL LIBRARY PREPARATION KIT MANUALPROTOCO LMANUAL PROCESSINGSTEP 1End Preparation
Starting Material
5 ng –1 μg fragmented DNA.
1.1

Mix the following components in a sterile nuclease-free tube:
iDeal Library End Repair/dA-Tailing Enzyme Mix (green)

3 µl

iDeal Library End Repair/dA-Tailing Buffer (green)

6.5 µlFragmented DNA

55.5 µl*

TOTAL volume

65 µl*If DNA Input is less than 55.5 μl, complete with water up to 55.5 μl.

1.2

Mix by pipetting followed by a quick spin to collect all liquid from thesides of the tube.

1.3

Place in a thermocycler, with the heated lid on, and run the following
program:• 30 minutes at 20°C
• 30 minutes at 65°C
• Hold at 4°C

8

IDEAL LIBRARY PREPARATION KIT MANUALPROTOCOLSTEP 2
Adaptor Ligation
NOTE: If DNA input is < 100 ng, dilute the iDeal Library Preparation Adaptor
for Illumina (provided at 15 μM) 1:10 in 10mM TRIS-HCl with 10 mM NaCl to a
final concentration of 1.5 μM. Use immediately.

2.1

Add the following components directly to the iDeal Library End Prep
reaction mixture and mix well:iDeal Library Ligation Master Mix (red)15 µliDeal Library Adaptor for Illumina* (red)2.5 µliDeal Library Ligation Enhancer (red)

1 µl

TOTAL volume

83.5 µl

2.2

Incubate at 20°C for 15 minutes in a thermal cycler.

2.3

Add 3 µl of iDeal Library Uracil Excision Reagent (red) to the ligation
mixture.

2.4

Mix well and incubate at 37°C for 15 minutes.
NOTE: If needed samples can be stored overnight at -20°C.

9

OPTION A
Size Selection of adaptor-ligated DNA
Size selection is optional. If the starting material is less than 50 ng, size
selection is not recommended. If you are not performing size selection,
proceed to the next page and perform clean-up step prior to PCR
amplification. The following size selection protocol is for libraries with200 bp inserts only. For libraries with different size fragment inserts,
refer to Table 4 for the appropriate volume of beads to be added. The size
selection protocol is based on a starting volume of 100 µl.
Table 4: Recommended conditions for bead based size selection.
Approximate insert size
Total Library size (insert + adaptor)

150 bp200 bp250 bp300 - 400 bp

400 - 500 bp

500 - 700 bp

270 bp320 bp400 bp400 - 500 bp

500 - 600 bp

600 - 800 bp

Volume of ligation reaction
Volume of beads
to add100 µl

1st Bead Selection

65 µl

55 µl

45 µl

40 µl

35 µl

30 µl

2nd Bead Selection

25 µl

25 µl

25 µl

20 µl

15 µl

15 µl

NOTE: If neede the libraries can be stored at -20°C.

10

2.5

Vortex AMPure XP beads to resuspend.

2.6

Add 13.5 µl dH2O to the ligation reaction for a 100 µl total volume.

2.7

Add 55 µl of resuspended AMPure XP beads to the 100 µl ligation
reaction. Mix well by pipetting up and down at least 10 times.

2.8

Incubate for 5 minutes at room temperature.

2.9

Quickly spin the tube and place the tube on the DiaMag02 magnetic rack (Cat. No. B04000001) to separate the beads from thesupernatant. After the solution is clear (about 5 minutes), carefullytransfer the supernatant containing your DNA to a new tube (Caution:
do not discard the supernatant). Discard the beads that contain theunwanted large fragments.

IDEAL LIBRARY PREPARATION KIT MANUAL

2.11 Quickly spin the tube and place it on the magnetic rack to separate
the beads from the supernatant. After the solution is clear (about
5 minutes), carefully remove and discard the supernatant that
contains unwanted DNA. Be careful not to disturb the beads that
contain the desired DNA targets (Caution: do not discard beads).

PROTOCOL

2.10 Add 25 µl resuspended AMPure XP beads to the supernatant, mix
well and incubate for 5 minutes at room temperature.

2.12 Add 200 µl of 80% freshly prepared ethanol to the tube while in themagnetic rack. Incubate at room temperature for 30 seconds, andthen carefully remove and discard the supernatant.
2.13 Repeat Step 2.12 once for a total of two washes.
2.14 Air the dry beads for 5 minutes while the tube is on the magnetic
rack with the lid open.
2.15 Elute the DNA target from the beads into 17 µl of 10 mM Tris-HCl
or 0.1 X Te, pH 8.0. Mix well on a vortex mixer or by pipetting up anddown. Incubate for at least 2 minutes at room temperature. Quickly
spin the tube and place it on a magnetic stand. After the solution
is clear (about 5 minutes), transfer 15 µl to a new PCR tube for
amplification.
NOTE: Be sure not to transfer any beads. Trace amounts of bead carry over may
affect the optimal performance of the polymerase used in the iDeal Library
PCR Master Mix in the subsequent PCR step.

11

OPTION B
Alternatively, Clean-up of adaptor-ligated DNA without
Size Selection
2.5

Vortex AMPure XP beads to resuspend.

2.6

Add 86.5 µl resuspended AMPure XP beads to the ligation reaction.
Mix well by pipetting up and down at least 10 times.

2.7

Incubate for 5 minutes at room temperature.

2.8

Quickly spin the tube and place it on the DiaMag02 - magnetic rack(Cat. No. B04000001) to separate beads from supernatant. After thesolution is clear (about 5 minutes), carefully remove and discard thesupernatant. Be careful not to disturb the beads that contain DNA
targets (Caution: do not discard beads).

2.9

Add 200 µl of 80% freshly prepared ethanol to the tube while in themagnetic rack. Incubate at room temperature for 30 seconds, andthen carefully remove and discard the supernatant.

2.10 Repeat Step 2.9 once, for a total of two washes.
2.11 Air the dry beads for 5 minutes while the tube is on the magnetic
stand with the lid open.
2.12 Elute the DNA target from the beads by adding 17 µl of 10 mM
Tris-HCl, pH 8.0 or 0.1X Te.
2.13 NOTE: Be sure not to transfer any beads. Trace amounts of beadcarry over may affect the optimal performance of the polymeraseused in the iDeal library PCR Master Mix in the subsequent PCR
step.

12

IDEAL LIBRARY PREPARATION KIT MANUAL

2.15 Quickly spin the tube and place it on the magnetic stand.
2.16 After the solution is clear (about 5 minutes), transfer 15 µl to a new
PCR tube for amplification.

PROTOCOL

2.14 Mix well by pipetting up and down, or on a vortex mixer. Incubate forat least 2 minutes at room temperature.

13

STEP 3
PCR amplification
3.1

Mix the following components in sterile strip tubes:
Adaptor Ligated DNA Fragments

15 µl

iDeal Library PCR Master Mix (blue)

25 µl

iDeal Library Index Primer* (blue)

5 µl

iDeal Universal PCR Primer* (blue)

5 µl

TOTAL volume

50 µl* These primers (Index Primer Set 1) are included. Index Primer Set 2 may bepurchased separately (Cat. No. C05010021).
NOTE: If needed the samples can be stored at-20°C.

PCR cycling conditions
3.2

Transfer tubes to a pre-programmed thermal cycler and incubate as
follows.
Cycle Step

Temp

Time

Cycles

Initial Denaturation

98°C

30 seconds

1

Denaturation

98°C

10 seconds

Annealing - Extension

65°C

75 seconds

Final Extension

65°C

5 minutes

Hold

4°C

∞

4 – 12*
1*Suggestion: 4 PCR cycles for 1 μg DNA input, 7 - 8 cycles for 50 ng,
and 12 for 5 ng DNA input. Further optimization of PCR cycle number
may be required.

14

IDEAL LIBRARY PREPARATION KIT MANUAL

3.3

Vortex AMPure XP beads to resuspend.

3.4

Add 45 µl of resuspended AMPure XP beads to the PCR reactions(~50 µl). Mix well by pipetting up and down at least 10 times.

3.5

Incubate for 5 minutes at room temperature.

3.6

Quickly spin the tube and place it on DiaMag02 - magnetic rack toseparate beads from supernatant. After the solution is clear (about
5 minutes), carefully remove and discard the supernatant. Be careful
not to disturb the beads that contain DNA targets (Caution: do notdiscard beads).

3.7

Add 200 µl of 80% ethanol to the tubes while in the DiaMag02 magnetic rack. Incubate at room temperature for 30 seconds, andthen carefully remove and discard the supernatant.

3.8

Repeat Step 3.7 once.

3.9

Air dry the beads for 5 minutes while the tubesare on the magnetic
stand with the lid open.

PROTOCOL

Clean-up of PCR amplification

3.10 Elute DNA target from beads into 33 µl of 0.1X TE. Mix well by
pipetting up and down at least 10 times. Incubate for at least 2
minutes at room temperature. Quickly spin the tube and place it on
an appropriate magnetic stand to separate beads from supernatant.
After the solution is clear (about 5 minutes), carefully transfer 30 µl
supernatant to a new PCR tube. Store libraries at –20°C.
3.11 Check the size distribution on an Agilent Bioanalyser High Sensitivity
DNA Chip. The sample may need to be diluted before loading. A 5
fold dilution can be used.

15

AUTO PR OTOCOL
AUTOMATED PROCESSING

The “Ideal_Library_Preparation” protocol on the iP-Star® Compact isusing the standard iDeal Library Preparation Kit from Diagenode. The
iDeal Library Preparation kit allows the preparation of indexed libraries
of genomic or ChIP DNA.

AUTO PROTOCOL

ProtocolIt provides flexibility to prepare 1 to 32 libraries in one run starting with
5 ng of DNA. The whole protocol takes approximately 1h30. It allows you
to prepare up to 96 libraries per day with 3 runs. At the end, you recoverligated products ready for amplification.

17

IP-Star setup
1.1

Switch ON the IP-Star Compact.

1.2

Select “Protocols” icon and then click on “Library prep”.

1.3

Under “Library prep”, select “Ideal_Library_Preparation”.
NOTE: If you plan to run between:-- 1 and 8 samples, chose “Ideal_Library_Preparation_08”-- 9 and 16 samples, chose “Ideal_Library_Preparation_16”-- 17 and 24 samples, chose “Ideal_Library_Preparation_24”-- 25 and 32 samples, chose “Ideal_Library_Preparation_32”

1.4

Setup the exact number of samples that you want to process.
NOTE: The Left Peltier Block is now cooling down to 4°C to keep the enzymes
and reagents cold.

1.5

Setup all the plastics on the platform according to the screen layout.

1.6

Fill TIP Rack 1 (and 2 if processing more than 8 samples) with tips
according to the screen.

TIP Rack 1

1.7

18

TIP Rack 2

12
11
10
9
8
7
6
5
4
3
2
1

Left Peltier
Block

12
11
10
9
8
7
6
5
4
3
2
1

Right Peltier
Block

Fill Left and Right Peltier Blocks with 200 µl tube strips according
to the screen.

IDEAL LIBRARY PREPARATION KIT MANUALEnd Preparation and Adaptor Ligation

AUTO PROTOCOLSTEP 1 & 2
NOTE: Allow the reagents from iDeal Library Preparation Kit to come at 4°C.
Work on ice from this point.

2.1

Prepare the following mixes.
• iDeal Library End Prep Mix:
Number of samples

1

8

16

24

32

iDeal Library End Repair/dA-Tailing
Enzyme Mix (green)

3 µl

24 µl

48 µl

72 µl

96 µl

iDeal Library End Repair/dA-Tailing
Buffer (green)

6.5 µl52 µl

104 µl156 µl

208 µlTOTAL

9.5 µl76 µl

152 µl

204 µl280 µl

NOTE: 55.5 µl of DNA will be added later for each sample.

• iDeal Library Adaptor Ligation Mix:
Number of samples

2.2

1

8

16

24

32

iDeal Library Ligation Master Mix (red)15 µl

120 µl240 µl360 µl

480 µliDeal Library Ligation Enhancer (red)

1 µl

8 µl

16 µl

24 µl

32 µl

TOTAL

16 µl

128 µl256 µl238 µl512 µlFill the strips of the Left Peltier Block according to the screen layout
with the following reagents:• Ligation Mix – 16 µl per well
• iDeal Library Adaptor for Illumina (red) – 2.5 µl per well
• iDeal Library Uracil Excision Reagent (red) – 3 µl per well

19

Left Peltier

Well 9-12• Uracil Excision Reagent

12
11
10
9
8
7
6
5
4
3
2
1
A B C

2.3

PCR tube

D E F G H

3 µl

Well 5-8• Adaptor for Illumina

2.5 µlWell 1-4
Adaptor Ligation Mix

16 µl• Ligation Master Mix

15 µl• Ligation Enhancer

1 µl

Fill the strips of the Right Peltier Block according to the screen
layout with:• iDeal Library End Prep Mix - 9.5 µl per well
• DNA sample – 55.5 µl per well
Right Peltier
12
11
10

Well 1-4
iDeal Library End Prep

65 µl

9
8
7

• Fragmented DNA

55.5 µl• End Repair/dA-Tailing Enzyme Mix• End Repair/dA-Tailing Buffer

6
5
4
3
2
1

Samples 25-32
Samples 17-24
Samples 9-16
Samples 1-8
A B C

2.4

20

PCR tube

D E F G H

Close the door and Run.

IDEAL LIBRARY PREPARATION KIT MANUAL

3 µl
6.5 µlSize Selection of adaptor-ligated DNA
Size selection is optional. If the starting material is less than 50 ng, size
selection is not recommended. If you are not performing size selection,
proceed to the clean-up step prior to PCR amplification.

AUTO PROTOCOLOPTION A

NOTE: Use the IP-Star and room temperature AMPure XP beads for the size
selection.

2.5

 Switch on the IP-Star and select “Protocols” icon and then “Libraryprep” category.

2.6

 Select “AMPure_XP_Size_Selection_08” if you plan to run between
1 and 8 samples, or “AMPure_XP_Size_Selection_16” if you plan torun between 9 and 16 samples.

2.7

 etup the exact number of samples that you want to process by
Spressing the black box.
NOTE: The Peltier Block 1 is now cooling down to 4°C to keep your samples cold.

Setup all the plastics on the platform according to the screen layout

12
11
10
9
8
7
6
5
4
3
2
1

G
H

D
E
F

1 2 3 4 5 696 plate 2C

7 8 9 10 11 12

Peltier
Block 2A
B

G
H

96 plate 1

C

Reagent
Rack 2D
E
F

Reagent
Rack 1

Peltier
Block 17 8 9 10 11 12

TIP Rack 2

1 2 3 4 5 6

TIP Rack 1

12
11
10
9
8
7
6
5
4
3
2
1

A
B

2.8

• F
 ill TIP Rack 1 (and 2 ifprocessing more than 8
samples) with tips according
to the screen.
• Fill Reagent Rack 1 & 2with reagent containers
according to the screen.
• Fill 96 plate 1 with a 96 well
microplate.
• Fill Peltier Block 1 with
200 µl tube strips according
to the screen.

21

2.9

Fill the robot with all reagents.
• Add 80 µl of ChIP-seq grade water to each sample to have a final
volume of 100 µl. Put your samples in lane 1 (and 2 if processing
more than 8 samples) of the Peltier Block 1.
• Distribute the AMPure XP beads according to the required size
following the recommendations from the table :AMPure XP

Final size

110 µl

250 bp

100 µl280 bp90 µl

325 bp80 µl

400 bp70 µl

500 bp

We recommend to use the size selection protocol for the final librarysize of 325 bp (insert + adaptor).
NOTE: Resuspend the beads with pipetting up and down several times before
dispense them.

• Fill the container of the Reagent Rack 1 with freshly prepared
80% Ethanol according to the screen.
• Fill the container of Reagent Rack 2 with Resuspension Buffer
according to the screen.
• Check the proper insertion of the racks and the consumables.
2.10 Close the door and press “Run” to start.
2.11 After the run, recover your samples on the upper row of the Left
Peltier Block. The final volume is 20 µl for each sample.
2.12 Press “OK” and “Back” until the homepage appears on the screen.
Press “Shutdown” and wait until the screen is black before switching
off the IP-Star.
NOTE: Remove all the plastics from the platform, empty the waste shuttle andclean the inner side of the IP-Star with 70% ethanol.

22

IDEAL LIBRARY PREPARATION KIT MANUALAlternatively, Clean-up of adaptor ligated DNA without
size selection
NOTE: Use the IP-Star and room temperature AMPure XP beads for the clean-up.

2.5

Select “Protocols” icon and then “IPure” category.

2.6

Select “AMPure_XP_Purification_08” if you plan to run between 1
and 8 samples, or “AMPure_XP_Purification_16” if you plan to run
between 9 and 16 samples.

2.7

Setup the exact number of samples that you want to process bypressing the black box.

AUTO PROTOCOLOPTION B

NOTE: The Peltier Block is now cooling down to 4°C to keep your samples cold.

Setup all the plastics on the platform according to the screen layout.
12
11
10
9
8
7
6
5
4
3
2
1

G
H

D
E
F

1 2 3 4 5 696 plate 2C

7 8 9 10 11 12

Peltier
Block 2A
B

G
H

96 plate 1

D
E
F

Reagent
Rack 27 8 9 10 11 12

Reagent
Rack 1

Peltier
Block 11 2 3 4 5 6

TIP Rack 2

C

TIP Rack 1

12
11
10
9
8
7
6
5
4
3
2
1

A
B

2.8

• Fill TIP Rack 1 (and 2 ifprocessing more than 8
samples) with tips according
to the screen.
• Fill Reagent Rack 1 & 2with reagent containers
according to the screen.
• Fill 96 plate 1 with a 96 well
microplate.
• Fill Peltier Block 1 with
200 μl tube strips according
to the screen.

23

2.9

Fill the robot with all reagents• Put your samples in lane 1 (and 2 if processing more than 8
samples) of the Peltier Block 1.
• Distribute 86.5 µl room temperature AMPure XP beads in eachwell of row 1 (and 5 if processing more than 8 samples) of the
96-well Microplate.
NOTE: Resuspend the beads with pipetting up and down several times before
dispense them.

• Fill the container of the Reagent Rack 1 with freshly prepared
80% Ethanol according to the screen.
• Fill the container of Reagent Rack 2 with Resuspension Buffer
according to the screen.
• Check the proper insertion of the racks and the consumables.
2.10 Close the door and press “Run” to start.
2.11 After the run, recover your samples on the upper row of the Peltier
Block 1. The final volume is 20 μl per each sample.
2.12 Press “OK” and “Back” until the homepage appears on the screen.
Press “Shutdown” and wait until the screen is black before switching
off the IP-Star.
NOTE: Remove all the plastics from the IP-Star platform, empty thewaste shuttle, and clean the inner side of the IP-Star with 70% ethanol.

24

IDEAL LIBRARY PREPARATION KIT MANUALAUTO PROTOCOLSTEP 3
PCR amplification
3.1

Mix the following components in sterile strip tubes:
Adaptor Ligated DNA Fragments

15 µl

iDeal Library PCR Master Mix (blue)

25 µl

iDeal Library Index Primer* (blue)

5 µl

iDeal Universal PCR Primer* (blue)

5 µl

TOTAL volume

50 µl* These primers (Index Primer Set 1) are included. Index Primer Set 2 may bepurchased separately (Cat. No. C05010021).

NOTE: If needed the samples can be stored at-20°C.

PCR cycling conditions
3.2

Transfer tubes to a pre-programmed thermal cycler and incubate as
follows.
Cycle Step

Temp

Time

Cycles

Initial Denaturation

98°C

30 seconds

1

Denaturation

98°C

10 seconds

Annealing - Extension

65°C

75 seconds

Final Extension

65°C

5 minutes

Hold

4°C

∞

4 – 12*
1*Suggestion: 4 PCR cycles for 1 μg DNA input, 7 - 8 cycles for 50 ng, and 12 for
5 ng DNA input. Further optimization of PCR cycle number may be required.

25

Clean-up of PCR amplification
NOTE: Use the IP-Star and room temperature AMPure XP beads for the clean-up.

3.3

Select “Protocols” icon and then “IPure” category.

3.4

Select “AMPure_XP_Purification_08” if you plan to run between 1
and 8 samples, or “AMPure_XP_Purification_16” if you plan to run
between 9 and 16 samples.

3.5

Setup the exact number of samples that you want to process bypressing the black box.
NOTE: The Peltier Block is now cooling down to 4°C to keep your samples cold.

3.6

Setup all the plastics on the platform according to the screen layout.
• Fill TIP Rack 1 (and 2 if processing more than 8 samples) withtips according to the screen.
• Fill Reagent Rack 1 & 2 with reagent containers according to thescreen.
• Fill 96 plate 1 with a 96 well microplate.
• Fill Peltier Block 1 with 200 μl tube strips according to thescreen.

3.7

Fill the robot with all reagents• Put your samples in lane 1 (and 2 if processing more than 8
samples) of the Peltier Block 1.
• Distribute 45 µl room temperature AMPure XP beads in each wellof row 1 (and 5 if processing more than 8 samples) of the
96-well Microplate.
NOTE: Resuspend the beads with pipetting up and down several times before
dispense them.

• Fill the container of the Reagent Rack 1 with freshly prepared
80% Ethanol according to the screen.
• Fill the container of Reagent Rack 2 with Resuspension Buffer
according to the screen.
26

IDEAL LIBRARY PREPARATION KIT MANUAL

3.8

Close the door and press “Run” to start.

3.9

After the run, recover your samples on the upper row of the Peltier
Block 1. The final volume is 20 μl per each sample.

3.10 Press “OK” and “Back” until the homepage appears on the screen.
Press “Shutdown” and wait until the screen is black before switching
off the IP-Star.

AUTO PROTOCOL• Check the proper insertion of the racks and the consumables.

NOTE: Remove all the plastics from the IP-Star platform, empty the waste
shuttle, and clean the inner side of the IP-Star with 70% ethanol.

27

Related products

28

Product

Cat. No.

iDeal ChIP-Seq Kit for Histones

C01010051

Auto iDeal ChIP-seq Kit for Histones

C01010171

Bioruptor Pico

B01060010

IP-Star Compact Automated System

B03000002

DiaMag 0.2 ml - magnetic rack

B04000001

MagMeDIP-seq kit

C02010023

Auto MagMeDIP-seq kit

C02010016

IDEAL LIBRARY PREPARATION KIT MANUALFOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
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29

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