Fluidigm Juno Getting Started Manual

Genotyping
with Juno

Getting Started Guide

PN 100-7074 D1

For Research Use Only. Not for use in diagnostic procedures.

Information in this publication is subject to change without notice. It is
Fluidigm policy to improve products as new techniques and components
become available. Therefore, Fluidigm reserves the right to change
specifications at any time. Every effort has been made to avoid errors in
the text, diagrams, illustrations, figures, and screen captures. However,
Fluidigm assumes no responsibility for any errors or omissions. In no
event shall Fluidigm be liable for any damages in connection with or
arising from the use of this publication.

Patent and Limited License Information

Fluidigm products are covered by issued and pending patents in the
United States and other countries. Patent and limited license information
is available at fluidigm.com/legalnotices

Limited Use License to Perform Preamplification with Fluidigm IFCs

A license to use Thermo Fisher Scientific’s patented preamplification
method workflows involving a Fluidigm integrated fluidic circuit (IFC) can
be obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation
or (ii) by a separate license from Thermo Fisher Scientific. For licensing
information, contact [e-mail].

Limited Digital PCR License

A license to use Thermo Fisher Scientific’s patented digital PCR method
in all fields other than in the Sequencing Field, the Mass Spectrometry
Field, and the Prenatal Field in workflows involving a Fluidigm IFC can be
obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation or
(ii) by a separate license from Thermo Fisher Scientific. For licensing
information, contact [e-mail].

Trademarks

Fluidigm, the Fluidigm logo, Biomark, EP1, Juno, and SNP Type are
trademarks or registered trademarks of Fluidigm Corporation in the
United States and/or other countries. All other trademarks are the sole
property of their respective owners.

For EU's WEEE directive information, go to fluidigm.com/compliance

© 2017 Fluidigm Corporation. All rights reserved. 09/2017

For technical support visit fluidigm.com/support.

North America [phone] | Toll-free (US/CAN): 866 358 4354 | [e-mail]
Latin America [phone] | [e-mail]
Europe/Middle East/Africa/Russia [phone] | [e-mail]
Japan [phone] | [e-mail]
China (excluding Hong Kong) [phone] | [e-mail]
All other Asian countries/India/Australia [phone] | [e-mail]

2

Genotyping with Juno Getting Started Guide

Contents

About This Guide 4

Purpose 4

How to Use This Guide 5

Safety Alert Conventions 5

Safety Alerts for Chemicals 5

Safety Alerts for Instruments 5

Safety Data Sheets 6

Getting Started 7

Workflow 7

Best Practices 7

Related Documentation 7

Chapter 1: Product Information 8

Required Kit Contents 8

TaqMan Assay Kit 8

SNP Type Assay Kit 9

Required Reagents 10

TaqMan Assays 10

SNP Type Assays 10

Suggested Reagents 10

Required Consumables 11

Required Equipment 11

Obtain the Minimum Required Genomic DNA 14

Prepare the Sample Mix 15

Load and Run the IFC on Juno 16

Perform Genotyping Analysis on the Samples 19

19

Chapter 3: Genotyping with the Juno

96.96 Genotyping IFC Using SNP
Type Assays 20

Prepare Assay and Sample Mixes 20

Prepare the 200 nM Primer Pool for
Preamplification 20

Prepare 2X SNP Type Assays 21

Prepare the Assay Mix 22

Obtain the Minimum Required Genomic DNA 22

Prepare the Sample Mix 23

Load and Run the IFC on Juno 24

Perform Genotyping Analysis on the Samples 27

27

Appendix A: Suggested Kits 28

TaqMan Assay Kit 28

SNP Type Assay Kit 28

Suggested Reagents to Use with TaqMan
Assay and SNP Type Assay Kits 29

Suggested Equipment 11

Required Software 12

IFC Type and Related Scripts 12

Chapter 2: Genotyping with the
Juno 96.96 Genotyping IFC Using
TaqMan Assays 13

Prepare Assay and Sample Mixes 13

Prepare the Primer Pool for Preamplification 13

Prepare 2X TaqMan Assays for Genotyping 14

Prepare the Assay Mix 14

Genotyping with Juno Getting Started Guide

Appendix B: Safety 30

General Safety 30

Instrument Safety 30

Electrical Safety 31

Chemical Safety 31

Disposal of Products 31

3

About This Guide

CAUTION ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard

types specified in procedures may be abbreviated in this document. For
complete safety information, see the safety appendix on page 30.

Purpose

This guide describes how to perform genotyping of low-concentration DNA with
the Juno™ 96.96 Genotyping IFC (integrated fluidic circuit) on the Juno™ system.
This is possible through advanced microfluidics technology that integrates
preamplification and genotyping reactions of up to 96 samples and 96
genotyping assays in a single workflow on an IFC.

The IFC produces 9,216 genotypes in less than three hours using a simple
workflow with minimal hands-on time. Samples are loaded into individual inlets of
the Juno 96.96 Genotyping IFC, then distributed across multiple reaction
chambers in nanoliter-volume aliquots. With high-quality samples, detecting the
specific targets requires thermal cycling for preamplification and PCR for
genotyping on the instrument.

After genotyping is performed on the Juno system, the IFC is scanned on the
EP1™ system or the Biomark™ HD system to collect genotyping data for later
analysis.

4

Genotyping with Juno Getting Started Guide

How to Use This Guide

The chapters in this guide are organized according to assay type. Refer to the
appropriate chapter to run the Juno 96.96 Genotyping IFC on the Juno system.

For detailed instructions on instrument and software operation, refer to the Juno
System User Guide (PN 100-7070).

Safety Alert Conventions

This guide uses specific conventions for presenting information that may require
your attention. Refer to the following safety alert conventions.

Safety Alerts for Chemicals

Fluidigm follows the United Nations Globally Harmonized System (GHS) for
communicating chemical hazard information. GHS provides a common means of
classifying chemical hazards and a standardized approach to chemical label
elements and safety data sheets (SDSs). Key elements include:

About This Guide

How to Use This Guide

• Pictograms that consist of a symbol on a white background within a red diamond­shaped frame. Refer to the individual SDS for the applicable pictograms and
warnings pertaining to the chemicals being used.

• Signal words that alert the user to a potential hazard and indicate the severity level.
The signal words used for chemical hazards under GHS:

DANGER Indicates more severe hazards.

WARNING Indicates less severe hazards.

Safety Alerts for Instruments

For hazards associated with instruments, this guide uses the following indicators:

• Pictograms that consist of a symbol on a white background within a black triangle­shaped frame.

Genotyping with Juno Getting Started Guide

5

About This Guide

Safety Data Sheets

• Signal words that alert the user to a potential hazard and indicate the severity level.
The signal words used for instrument hazards:

DANGER Indicates an imminent hazard that will result in severe injury or death if not
avoided.

WARNING Indicates a potentially hazardous situation that could result in serious
injury or death.

CAUTION Indicates a potentially hazardous situation that could result in minor or
moderate personal injury.

IMPORTANT Indicates information necessary for proper use of products or
successful outcome of experiments.

Safety Data Sheets

Read and understand the SDSs before handling chemicals. To obtain SDSs for
chemicals ordered from Fluidigm Corporation, either alone or as part of this system,
go to fluidigm.com/sds and search for the SDS using either the product name or the
part number.

Some chemicals referred to in this user guide may not have been provided with your
system. Obtain the SDSs for chemicals provided by other manufacturers from those
manufacturers.

6

Genotyping with Juno Getting Started Guide

Getting Started

Workflow

Reagent Handling Automated Steps Estimated Times

1 Prepare preamplification and

genotyping assay and sample mixes

2 Pipet preamplification, genotyping

mixes, and control line fluid into the IFC

3

4

Run a script to preamplify and
genotype the DNA

Perform genotyping analysis on
EP1 or Biomark systems

30–60 minutes

10–20 minutes

2.5 hours (TaqMan
protocol); 3.5 hours
(SNP Type protocol)

5–10 minutes

Best Practices

• Use good laboratory practices to minimize contamination of samples. Use a new

pipette tip for every new sample. Whenever possible, separate pre- and post­PCR activities. Dedicate laboratory materials to designated areas.

• Unless otherwise specified, thaw reagents at room temperature, then use them
at room temperature. Store reagents at their specified storage temperatures.
(See “Required Kit Contents” on page 8.)

• Vortex reagents for 20 seconds, and then centrifuge reagents for 2 seconds
before use.

Related Documentation

Go to fluidigm.com/documents

Genotyping with Juno Getting Started Guide

7

Chapter 1: Product Information

Required Kit Contents

The kits include the reagents required for preparing 10 IFCs to use on the Juno
system. For suggested kits, see “Suggested Kits” on page 28.

IMPORTANT

• Do not pipet reagents from the TaqMan and SNPType assay kits into the same IFC.

Use a different IFC for each kit. Do not mix reagents from different kits.

• Unless otherwise specified, thaw reagents at room temperature, then use them at

room temperature. Store reagents at their specified storage temperatures. Vortex
reagents for 20 seconds, then centrifuge reagents for 2 seconds before use.

TaqMan Assay Kit

Box Component Cap Color Quantity Volume

per Tube (mL)

Juno Genotyping Kit
for 10 IFCs
(PN 100-8362)

Juno GT Flux Fluid

Juno GT Preamp
Master Mix

Dilution Reagent Natural 2 tubes 1.7

Probe GT Master Mix Gold 2 tubes 1.6

Juno GT Flux Fluid Purple 1 tube 0.9

Juno 96.96
Genotyping IFC—
10 IFCs

Juno 96.96 GT
Control Line Fluid

Light
purple

— 10 IFCs —

— 2 boxes;

Probe GT Master Mix

Juno GT Preamp Master Mix

Dilution Reagent

1 tube 1.35

20 syringes/
box

—

Storage

–20 ºC

Room
temperature

Figure 1. Juno™ Genotyping Kit for 10 IFCs (PN 100-8362).

8

Genotyping with Juno Getting Started Guide

SNP Type Assay Kit

Chapter 1: Product Information

Required Kit Contents

Box Component Cap Color Quantity Volume

per Tube
or Bottle

Juno SNP Type
Genotyping Kit for
10 IFCs
(PN 100-8364)

Juno GT Preamp Master
Mix

Juno SNP Type GT
Master Mix

60X SNP Type Reagent Amber

Light

1 tube 1.35 mL

purple

Light

2 tubes 1.6 mL

blue

2 tubes 70 μL

Juno GT Flux Fluid Purple 1 tube 1.0 mL

Dilution Reagent Natural • 2 bottles

• 1 tube

Juno 96.96 Genotyping

— 10 IFCs —

• 3.7 mL

• 1.7 mL

IFC—10 IFCs

Juno 96.96 GT Control
Line Fluid

— 2 boxes;

20 syringes/

—

box

Juno GT Flux Fluid

60X SNP Type Reagent

Storage

–20 ºC

Room
temperature

Dilution Reagent

Juno SNP Type GT Master Mix

Juno GT Preamp Master Mix

Figure 2. Juno™ SNP Type Genotyping Kit for 10 IFCs (PN 100-8364).

Genotyping with Juno Getting Started Guide

9

Chapter 1: Product Information

Required Reagents

Required Reagents

TaqMan Assays

Product Name Company Part Number

20X, 40X, or 80X TaqMan
assays

®

genotyping

Thermo Fisher
Scientific

—

SNP Type Assays

Product Name Company Part Number

SNP Type assays specific target
amplification Primers (100 μM STA)

SNP Type assays ASP1/ASP2
(100 μM each)

SNP Type assays LSP
(100 μM each)

Fluidigm —

Fluidigm —

Fluidigm —

Suggested Reagents

Product Name Company Part Number

UltraPure
Water

™

DNase/RNase-Free Distilled

Thermo Fisher
Scientific

10977-015

10

Genotyping with Juno Getting Started Guide

Chapter 1: Product Information

Required Consumables

Product Name Company Part Number

Juno 96.96 Genotyping IFC:

• Juno 96.96 Genotyping IFC

• Juno 96.96 Genotyping IFC, 10 Pack

Fluidigm 100-6499

100-8365

Required Consumables

Disposable microcentrifuge tubes,
polypropylene, 1.5 mL

96-well PCR plates MLS†

MicroAmp® Clear Adhesive Film Thermo Fisher

* Recommended: VWR® Slick Disposable Microcentrifuge Tubes, Polypropylene, 1.5 mL

(VWR PN 20170-666)

† Recommended: TempPlate® semi-skirted 96-well PCR plates (USA Scientific

PN 1402-9700)

Major laboratory
supplier (MLS)*

Scientific

—

—

4306311

Required Equipment

Product Name Company Part Number

Juno system, including system software version v3.1 or
later, instrument, software, MX Interface Plate, Interface
Plate Loading Fixture, Cleaning Plate, and Barrier Tape
Applicator and Adapter

For Juno 96.96 Genotyping IFC: SX Interface Plate Fluidigm 100-6368

Fluidigm 101-6455

Vortexer MLS —

Pipettes (P2, P20, P200, P1000) and appropriate
low-retention tips

8-channel pipettes and appropriate low-retention tips MLS —

Microcentrifuge MLS —

Suggested Equipment

Product Name Company Part Number

Two biocontainment hoods (DNA hood and DNA-free hood)
to prevent DNA contamination of lab and samples

Genotyping with Juno Getting Started Guide

MLS —

MLS —

11

Chapter 1: Product Information

Required Software

Required Software

• Fluidigm Data Collection software v4.2 or later

• Fluidigm SNP Genotyping Analysis software v4.2 or later

IFC Type and Related Scripts

Barcode (prefix) Scripts Description

180x Juno 96.96 Fast Preamplification and genotyping of samples by

TaqMan assays (180x)

180x Juno 96.96 Preamplification and genotyping of samples by SNP

Type assay (180x).

12

Genotyping with Juno Getting Started Guide

Chapter 2: Genotyping with the
Juno 96.96 Genotyping IFC Using
TaqMan Assays

Prepare Assay and Sample Mixes

Prepare the Primer Pool for Preamplification

1 If necessary, adjust the concentration of TaqMan genotyping assays with DNase-free

water to 18 μM (20X).

2 In a new, labeled 1.5-mL microcentrifuge tube, combine 2 μL of each 20X TaqMan

genotyping assay up to a total of 96 assays. The total volume of assays is 2Y in
Table 1, where Y is the number of assays used. Each assay is at a final concentration
of 0.2X in the primer pool.

3 Add Dilution Reagent to the 20X TaqMan assays:

Table 1. Prepare the primer pool for preamplification

Component Volume (μL) Final Concentration

20X TaqMan genotyping assays,
18 μM*

Dilution Reagent 200 – 2Y —

Total 200.0 —

* See step 1.

The final concentration of each primer in the preamplification reaction is 45 nM.

NOTE The volume can be adjusted proportionally based on the number of samples

to be amplified.

2Y (up to 96 assays) 180 nM (0.2X)

Genotyping with Juno Getting Started Guide

13

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Prepare Assay and Sample Mixes

Prepare 2X TaqMan Assays for Genotyping

1 If necessary, adjust the concentration of TaqMan genotyping assays with DNase-free

water to 18 μM (20X).

2 In a new 96-well plate, dilute the 20X TaqMan genotyping assays in Dilution

Reagent or DNase-free water to a final concentration of 2X for each assay:

Component Volume (μL) Final Concentration

20X TaqMan genotyping assays 1.0 2X

Dilution Reagent or
DNase-free water

Total 10.0 —

9.0 —

Prepare the Assay Mix

1 Label a new 96-well plate, “TAQMAN ASSAY PLATE.” In a DNA-free hood, pipet

2.5 μL of Probe GT Master Mix into each well. (See Table 2.)

2 In a DNA-free hood, pipet 2.5 μL of 2X TaqMan assays into a well of the TaqMan

assay plate for each assay. (See “Prepare 2X TaqMan Assays for Genotyping”.)

3 In unused assay inlets, combine 2.5 μL of Probe GT Master Mix with 2.5 μL DNase-

free water.

4 Seal the plate with MicroAmp Clear Adhesive Film, vortex it for 5 seconds, then

centrifuge it at 1,000 x g for 1 minute.

Table 2. Assay mix

Component Volume (μL)

Probe GT Master Mix 2.5

2X TaqMan assays*

Total 5.0

* See “Prepare 2X TaqMan Assays for Genotyping”.

Obtain the Minimum Required Genomic DNA

For high-quality human samples, the minimum DNA required is 2.5 ng/μL in 2.75 μL.
Larger genomes require higher concentrations of genomic DNA.

14

2.5

Genotyping with Juno Getting Started Guide

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Prepare Assay and Sample Mixes

Prepare the Sample Mix

1 In a DNA-free hood, in a new 1.5-mL microcentrifuge tube labeled “Sample Pre-Mix,”

combine the Juno GT Preamp Master Mix and the primer pool for preamplification to
prepare the sample pre-mix. (See Table 3.)

2 Label a new 96-well plate “SAMPLE PLATE.” Pipet 2.25 μL of the sample pre-mix

into each well of the plate. Skip wells that are for no template controls. Do not add
sample pre-mix to no template control wells.

IMPORTANT Prepare at least one no template control.

3 In a DNA sample hood, pipet 2.75 μL of genomic DNA into the appropriate wells of

the sample plate.

4 In a DNA sample hood, pipet 5.00 μL of Dilution Reagent into each no template

control well.

5 Seal the plate with MicroAmp Clear Adhesive Film, vortex it for 5 seconds, then

centrifuge it at 1,000 x g for 1 minute.

Table 3. Sample mix

Component Volume per

Inlet (μL)

SAMPLE PRE-MIX

Juno GT Preamp Master Mix

Primer pool for preamplification†

Genomic DNA

Total

* 120 reactions for ease of pipetting
† See “Prepare the Primer Pool for Preamplification” on page 13.

Volume per

Inlet with

Overage

0.8 1.00 120.0

1.0 1.25 150.0

2.2 2.75 —

4.0 5.0 270.0

(μL)

Sample Mix
for IFC with

Overage*

(μL)

IMPORTANT Do not go past the first stop on the pipette. Doing so may introduce air

bubbles into the inlets. To avoid bubbles, pipet 4.0 μL into each inlet from the 5.0 μL
overage volume.

Genotyping with Juno Getting Started Guide

15

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Load and Run the IFC on Juno

Load and Run the IFC on Juno

1 Review the loading map, which is affixed to the bottom of every new Juno 96.96

Genotyping IFC. The loading map is a general guide to show you how to pipet
samples, assays, flux fluid, and control line fluid:

A1

16

Genotyping with Juno Getting Started Guide

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Load and Run the IFC on Juno

2 Review the pipetting map, which provides specific instructions for pipetting reagents

in the IFC. Pipet reagents from the TaqMan assay plate and the sample plate to the
IFC. On the pipetting map, each inlet is labeled with the plate well location of the
sample or assay to be pipetted into that inlet:

A1

A01

A02 A03 A04 A05

A07

A08 A09 A10 A11

B01

B02 B03 B04 B05

B07

B08 B09 B10 B11

C01

C02 C03 C04 C05

C07 C08 C09 C10 C11

D01 D02 D03 D04 D05

D07 D08 D09 D10 D11

E01

E02 E03 E04 E05 E06

E07

E08 E09 E10 E11 E12

F01 F02 F03

F04 F05

F07 F08 F09 F10 F11

G01

G02 G03 G04 G05

G07

G08 G09 G10 G11

H01

H02 H03

H04 H05

H07

H08 H09

H10 H11

A06

A12

B06

B12

C06

C12

D06

D12

F06

F12

G06

G12

H06

H12

Acc 1

Acc 2

A01

A02 A03 A04 A05

A07

A08 A09 A10 A11

B01

B02 B03 B04 B05

B07

B08 B09 B10 B11

C01

C02 C03 C04 C05

C07 C08 C09 C10 C11

D01 D02 D03 D04 D05

D07 D08 D09 D10 D11

vv

E01

E02 E03 E04 E05 E06

E07

E08 E09 E10 E11 E12

F01 F02 F03

F07 F08 F09 F10 F11

G01

G02 G03 G04 G05

G07

G08 G09 G10 G11

H01

H02 H03

H07

H08 H09

Key

Load 1 Load 2

Juno 96.96 GT Control
Line Fluid

Juno GT Flux Fluid,
15 μL

F04 F05

H04 H05

H10 H11

A06

A12

B06

B12

C06

C12

D06

D12

F06

F12

G06

G12

H06

H12

Assay mix, 4.0 μL

Sample mix, 4.0 μL

Juno 96.96 GT Control
Line Fluid

—

Empty

Figure 1. Pipetting map for the Juno 96.96 Genotyping IFC

3 Ensure that the notched corner of the IFC (“A1”) is at the top left.

4 Load an entire syringe of Juno 96.96 GT Control Line Fluid in Acc1 and a second

syringe in Acc2. (See pink squares on the pipetting map.) To ensure correct
accumulator volume, only use syringes containing Juno 96.96 GT Control Line Fluid.

Genotyping with Juno Getting Started Guide

17

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Load and Run the IFC on Juno

5 Load an entire syringe of Juno 96.96 GT Control Line Fluid into a reservoir and a

second syringe into the second reservoir. (See long pink rectangles on the right side
of the pipetting map.)

IMPORTANT Carefully dispense control line fluid into the reservoirs. If control line

fluid comes into contact with the sample inlets, use a new IFC.

6 Pipet 15 μL of Juno GT Flux Fluid into each of the six ports. (See purple circles on the

pipetting map.)

7 Unseal the TaqMan assay plate and pipet 4.0 μL of each assay mix into an assay

inlet. (See black circles on the pipetting map and “Prepare the Assay Mix” on
page 14.)

8 Unseal the sample plate and pipet 4.0 μL of each sample mix into a sample inlet.

(See green circles on the pipetting map and “Prepare the Sample Mix” on page 15.)

IMPORTANT Do not go past the first stop on the pipette. Doing so may introduce air

bubbles into the inlets. Pipet 4.0 μL from the 5.0 μL overage volume to ensure that
no air bubbles enter the inlet.

9 Pull the sticker front tab down and away from the IFC to gently peel off the loading

map. Do not invert the IFC.

10 If necessary, remove any bubbles from an IFC inlet by removing the contents by

pipette and then carefully re-pipetting the contents into the inlet.

11 Ensure that the SX interface plate (silver label) is installed in the instrument. [See the

Juno System User Guide (PN 100-7070).]

12 Place the IFC into the Juno instrument, then start the run <60 minutes after pipetting

the reagents into the IFC.

13 On the Juno Scripts screen, tap the Probe GT tab, Juno 96.96 Fast, then Run. It

takes ~2.7 hours to complete.

The script contains these thermal cycling protocols:

Cycles Temperature Time

Hot start 95 ºC 2 min

14 95 ºC 15 sec

60 ºC 4 min

Cycles Temperature Time

Hot start 95 ºC 2 min

45 95 ºC 2 sec

18

60 ºC 20 sec

Genotyping with Juno Getting Started Guide

Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays

Perform Genotyping Analysis on the Samples

14 After the run is finished, tap EJECT to eject the IFC.

IMPORTANT After a run, perform an end-point read of the IFC in ≤1 hour. Do not

leave the IFC overnight in the instrument. Doing so will adversely affect the reaction.

Perform Genotyping Analysis on the
Samples

Refer to the appropriate document:

• SNP Genotyping User Guide (PN 68000098)

• Biomark HD Data Collection User Guide (PN 100-2451)

• Biomark/EP1 Data Collection User Guide (PN 68000127).

Genotyping with Juno Getting Started Guide

19

Chapter 3: Genotyping with the Juno

96.96 Genotyping IFC Using SNP Type
Assays

Prepare Assay and Sample Mixes

Prepare the 200 nM Primer Pool for Preamplification

1 In a new 1.5-mL microcentrifuge tube, combine 2 μL of 100 μM SNP Type assays

specific target amplification primers (100 μM STA) up to a total of 96 assays. The total
volume is Y in Table 4.

2 In the same microcentrifuge tube, combine 2 μL of 100 μM SNP Type assays locus-

specific primers (100 μM LSP) up to a total of 96 assays. The total volume is Z in
Table 4.

3 Add Dilution Reagent to the SNP Type assays:

Table 4. Pool SNP Type assays

Component Volume (μL) Final Concentration

(nM*)

SNP Type assays specific target amplification
primers (100 μM STA)

SNP Type assays locus-specific primers
(100 μM LSP)

Dilution Reagent 1,000 – (Y + Z)—

Total 1,000.0 —

* The final concentration of each primer in the preamplification reaction is 50 nM.

NOTE

• Volume can be adjusted proportionally based on the number of

samples to be amplified.

• You can store the pooled SNP Type STA assays at –20 °C for 1 year or ≤10

freeze-thaw cycles, whichever is shorter.

Y (up to 96 assays) 200.0

Z (up to 96 assays) 200.0

20

Genotyping with Juno Getting Started Guide

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Prepare Assay and Sample Mixes

Prepare 2X SNP Type Assays

Prepare 50X Primer Mix for Each Single Assay Inlet

In a DNA-free hood, in a new 96-well plate, combine the following reagents for each
assay:

Component Volume per 40 μL

Stock (μL)

SNP Type assays allele-specific primers
pooled ASP1 and ASP2 Primers (100 μM
ASP1/100 μM ASP2)

SNP Type assays locus-specific primers
(100 μM LSP)

Dilution Reagent 29.0 —

Total 40.0*

* A 40.0-μL volume is sufficient for 40, 2X SNP Type assays.

Final Concentration

(μM)

3.0 7.5

8.0 20.0

Prepare 2X SNP Type Assays from the 50X Primer Mix for
Genotyping

In a DNA-free hood, in a new 96-well plate, combine the following reagents for each
assay:

Component Volume per 25 μL

Stock (μL)

Concentration

Final

—

50X Primer Mix*

Dilution Reagent 24.0 —

Total 25.0†

* See “Prepare 50X Primer Mix for Each Single Assay Inlet”.
† A 25.0-μL volume is sufficient for 10 IFC runs.

1.0 2X

NOTE You can store the 2X SNP Type assays at –20 °C for up to one week.

Genotyping with Juno Getting Started Guide

—

21

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Prepare Assay and Sample Mixes

Prepare the Assay Mix

1 In a DNA-free hood, in a new 1.5-mL microcentrifuge tube labeled “Assay Pre-Mix,”

combine the Juno SNP Type GT Master Mix and 60X SNP Type Reagent to prepare
the assay pre-mix. (See Table 5.)

2 Label a new 96-well plate “SNP TYPE ASSAY PLATE.” In a DNA-free hood, pipet

2.5 μL of the assay pre-mix into each well.

3 Pipet 2.5 μL of 2X SNP Type assay into each well of the SNP Type assay plate.

4 In unused assay or no-assay control inlets, combine 2.5 μL of assay pre-mix with

2.5 μL of Dilution Reagent.

5 Seal the plate with MicroAmp Clear Adhesive Film, vortex it for 5 seconds, then

centrifuge it at 1,000 x g for 1 minute.

Table 5 Assay mix

Component Volume per

Inlet (μL)

ASSAY PRE-MIX

Juno SNP Type GT Master Mix

60X SNP Type Reagent

2X SNP Type assays†

Total

* 120 reactions for ease of pipetting
† See “Prepare 2X SNP Type Assays” on page 21.

1.933 2.417 290.0

0.066 0.083 10.00

2.00 2.5 —

4.00 5.00 300

Volume per

Inlet with

Overage

(μL)

Assay Mix

for IFC with

Overage*

Obtain the Minimum Required Genomic DNA

For high-quality human samples, the minimum DNA required is 2.5 ng/μL. Larger
genomes require higher concentrations of genomic DNA.

(μL)

22

Genotyping with Juno Getting Started Guide

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Prepare Assay and Sample Mixes

Prepare the Sample Mix

1 In a DNA-free hood, in a new microcentrifuge tube labeled “Sample Pre-Mix,”

combine the Juno GT Preamp Master Mix and the primer pool for preamplification to
prepare the sample pre-mix. (See Table 6.)

2 Label a new 96-well plate “SAMPLE PLATE,” and then pipet 2.25 μL of the sample

pre-mix into each well of the plate. Do not add sample pre-mix to no template control
wells.

IMPORTANT Prepare at least one no template control.

3 In a DNA sample hood, pipet 2.75 μL of genomic DNA into the appropriate wells of

the sample plate.

4 In a DNA sample hood, pipet 5.00 μL of Dilution Reagent into each no template

control well.

5 Seal the plate with MicroAmp Clear Adhesive Film, vortex it for 5 seconds, then

centrifuge it at 1,000 x g for 1 minute:

Table 6 Sample mix

Component Volume per

Inlet (μL)

SAMPLE PRE-MIX

Juno GT Preamp Master Mix

Primer pool for preamplification†

Genomic DNA

Total

* 120 reactions for ease of pipetting
† See “Prepare the 200 nM Primer Pool for Preamplification” on page 20.

0.800 1.00 120

1.00 1.25 150

2.20 2.75 —

4.00 5.00 270

Volume per

Inlet with

Overage

(μL)

Sample Mix for IFC

with Overage* (μL)

IMPORTANT Do not go past the first stop on the pipette. Doing so may introduce air

bubbles into the inlets. To avoid bubbles, pipet

4.0 μL into each inlet from the 5.0 μL overage volume.

Genotyping with Juno Getting Started Guide

23

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Load and Run the IFC on Juno

Load and Run the IFC on Juno

1 Review the loading map, which is affixed to the bottom of every new Juno 96.96

Genotyping IFC. The loading map is a general guide to show you how to pipet
samples, assays, and control line fluid:

A1

24

Genotyping with Juno Getting Started Guide

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Load and Run the IFC on Juno

2 Review the pipetting map, which provides specific instructions for pipetting reagents

in the IFC. Pipet reagents from the SNP Type assay plate and the sample plate to the
IFC. On the pipetting map, each inlet is labeled with the plate well location of the
sample or assay to be pipetted into that inlet:

A1

A01

A02 A03 A04 A05

A07

A08 A09 A10 A11

B01

B02 B03 B04 B05

B07

B08 B09 B10 B11

C01

C02 C03 C04 C05

C07 C08 C09 C10 C11

D01 D02 D03 D04 D05

D07 D08 D09 D10 D11

E01

E02 E03 E04 E05 E06

E07

E08 E09 E10 E11 E12

F01 F02 F03

F04 F05

F07 F08 F09 F10 F11

G01

G02 G03 G04 G05

G07

G08 G09 G10 G11

H01

H02 H03

H04 H05

H07

H08 H09

H10 H11

A06

A12

B06

B12

C06

C12

D06

D12

F06

F12

G06

G12

H06

H12

Acc 1

Acc 2

A01

A02 A03 A04 A05

A07

A08 A09 A10 A11

B01

B02 B03 B04 B05

B07

B08 B09 B10 B11

C01

C02 C03 C04 C05

C07 C08 C09 C10 C11

D01 D02 D03 D04 D05

D07 D08 D09 D10 D11

vv

E01

E02 E03 E04 E05 E06

E07

E08 E09 E10 E11 E12

F01 F02 F03

F07 F08 F09 F10 F11

G01

G02 G03 G04 G05

G07

G08 G09 G10 G11

H01

H02 H03

H07

H08 H09

Key

Load 1 Load 2

Juno 96.96 GT Control
Line Fluid

Juno GT Flux Fluid,
15 μL

F04 F05

H04 H05

H10 H11

A06

A12

B06

B12

C06

C12

D06

D12

F06

F12

G06

G12

H06

H12

Assay mix, 4.0 μL

Sample mix, 4.0 μL

Juno 96.96 GT Control
Line Fluid

—

Empty

3 Ensure that the notched corner of the IFC (“A1”) is at the top left.

4 Load an entire syringe of Juno 96.96 GT Control Line Fluid in Acc1 and a second

syringe in Acc2. (See the pink squares on the pipetting map.) To ensure correct
accumulator volume, only use syringes containing Juno 96.96 GT Control Line Fluid.

5 Load an entire syringe of Juno 96.96 GT Control Line Fluid into each of the two

reservoirs. (See the long pink rectangles on the right side of the pipetting map.)

IMPORTANT Carefully dispense control line fluid into the reservoirs. If control line

fluid comes into contact with the sample inlets, use a new IFC.

6 Pipet 15 μL of Juno GT Flux Fluid into each of the six ports. (See the purple circles on

the pipetting map.)

Genotyping with Juno Getting Started Guide

25

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Load and Run the IFC on Juno

7 Unseal the SNP Type assay plate and pipet 4.0 μL of each assay mix into an assay

inlet. (See the black circles on the pipetting map and “Prepare the Assay Mix” on
page 22.)

8 Unseal the sample plate and pipet 4.0 μL of each sample mix into a sample inlet.

(See the green circles on the pipetting map and “Prepare the Sample Mix” on
page 23.)

IMPORTANT Do not go past the first stop on the pipette. Doing so may introduce air

bubbles into the inlets. Pipet 4.0 μL from the 5.0 μL overage volume to ensure that
no air bubbles enter the inlet.

9 Pull the sticker front tab down and away from the IFC to gently peel off the loading

map. Do not invert the IFC.

10 If necessary, remove any bubbles from an IFC inlet by removing the contents by

pipette and then carefully re-pipetting the contents into the inlet.

11 Ensure that the SX interface plate (silver label) is installed in the instrument. [See the

Juno System User Guide (PN 100-7070).]

12 Place the IFC into the Juno instrument, then start the run <60 minutes after pipetting

the reagents into the IFC.

13 On Juno Scripts screen, tap the SNP Type tab, Juno 96.96, then Run. It takes

3 hours and 20 minutes to complete.

The script contains these thermal cycling protocols:

Table 7 Multiplex STA

Cycles Temperature Time

Hot start 95 ºC 2 min

14 95 ºC 15 sec

60 ºC 4 min

26

Genotyping with Juno Getting Started Guide

Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays

Table 8 SNP Type genotyping

Cycles Temperature Time

Hot start 95 ºC 10 min

95 ºC 15 sec

Perform Genotyping Analysis on the Samples

1

1

1

1

39

64 ºC 45 sec

72 ºC 15 sec

95 ºC 15 sec

63 ºC 45 sec

72 ºC 15 sec

95 ºC 15 sec

62 ºC 45 sec

72 ºC 15 sec

95 ºC 15 sec

61 ºC 45 sec

72 ºC 15 sec

95 ºC 15 sec

60 ºC 45 sec

72 ºC 15 sec

14 After the run is finished, tap EJECT to eject the IFC from the instrument.

IMPORTANT After a run, do not leave the IFC overnight in the instrument. Doing so

will adversely affect the reaction.

Perform Genotyping Analysis on the
Samples

Refer to the appropriate document:

• SNP Genotyping User Guide (PN 68000098)

• Biomark HD Data Collection User Guide (PN 100-2451)

• Biomark/EP1 Data Collection User Guide (PN 68000127).

Genotyping with Juno Getting Started Guide

27

Appendix A: Suggested Kits

Reagents and IFCs are available separately.

IMPORTANT Unless otherwise specified, thaw reagents at room temperature, then

use them at room temperature. Store reagents at their specified storage
temperatures. Vortex reagents for 20 seconds, then centrifuge reagents for
2 seconds before use.

TaqMan Assay Kit

Box Component Cap Color Quantity Volume

per Tube
or Bottle
(mL)

Juno Genotyping
Reagent Kit for
10 IFCs
(PN 100-8361)

Juno GT Preamp Master Mix Light

purple

Dilution Reagent Natural 2 tubes 1.7

Probe GT Master Mix Gold 2 tubes 1.6

Juno GT Flux Fluid Purple 1 tube 0.9

1 tube 1.35

SNP Type Assay Kit

Box Component Cap Color Quantity Volume

per Tube
or Bottle

Juno SNP Type
Genotyping
Reagent Kit
(PN 100-8363)

Juno GT Preamp Master Mix Light

purple

Juno SNP Type GT Master
Mix

60X SNP Type Reagent Amber

Juno GT Flux Fluid Purple 1 tube 1.0 mL

Light
blue

2 tubes 70 μL

1 tube 1.35 mL

2 tubes 1.6 mL

Storage

–20 ºC

Storage

–20 ºC

Dilution Reagent Natural • 2 bottles

28

• 3.7 mL

• 1 tube

Genotyping with Juno Getting Started Guide

• 1.7 mL

Suggested Reagents to Use with TaqMan Assay
and SNP Type Assay Kits

Appendix A: Suggested Kits

Box Component Cap Color Quantity Volume

per Tube
or Bottle

(mL)

Dilution Reagent
(PN 100-8726)

Juno GT IFC and
Control Line Fluid
Kit
(PN 100-8583)

Juno 96.96
Genotyping IFC
(PN 100-8365)

Juno 96.96
Genotyping IFC
(PN 100-6499)

Juno 96.96 GT
Control Line Fluid
(PN 100-8574)

Dilution Reagent Natural 1 bottle 25

Juno 96.96 Genotyping
IFC—10 IFCs

Juno 96.96 GT Control Line
Fluid

Juno 96.96 Genotyping IFC — 10 IFCs —

Juno 96.96 Genotyping IFC — 1 IFC —

Juno 96.96 GT Control Line
Fluid

— 10 IFCs —

— 2 boxes;

20 syringes/
box

— 20 syringes —

—

Storage

–20 ºC

Room
temperature

Room
temperature

Room
temperature

Room
temperature

Genotyping with Juno Getting Started Guide

29

Appendix B: Safety

General Safety

In addition to your site-specific safety requirements, Fluidigm recommends the
following general safety guidelines in all laboratory and manufacturing areas:

• Use personal protective equipment (PPE): safety glasses, fully enclosed shoes, lab
coats, and gloves.

• Know the locations of all safety equipment (fire extinguishers, spill kits, eyewashes/
showers, first-aid kits, safety data sheets, etc.), emergency exit locations, and
emergency/injury reporting procedures.

• Do not eat, drink, or smoke in lab areas.

• Maintain clean work areas.

• Wash hands before leaving the lab.

Instrument Safety

WARNING Do not modify this device. Unauthorized modifications may

create a safety hazard.

CAUTION HOT SURFACE The Juno thermal cycler chuck gets hot and can
burn your skin. Use caution when working near the chuck.

CAUTION PINCH HAZARD. The Juno door and tray can pinch your hand.
Make sure your fingers, hand, shirtsleeves are clear of the door and tray
when loading or ejecting an integrated fluidic circuit (IFC).

WARNING BIOHAZARD. If you are putting biohazardous material on the
instrument, use appropriate personal protective equipment and adhere to
Biosafety in Microbiological and Biomedical Laboratories (BMBL) from the
Centers for Disease Control and Prevention and to your lab’s safety
protocol to limit biohazard risks. If biohazardous materials are used,
properly label the equipment as a biohazard. For more information, see the
BMBL guidelines at: cdc.gov/biosafety/publications/index.htm

For a full list of the symbols on the instrument, refer to the Juno System User
Guide (PN 100-7070).

30

Genotyping with Juno Getting Started Guide

Electrical Safety

WARNING ELECTRICAL HAZARD. Electrical shock can result if the Juno

instrument is operated without its protective covers.

WARNING ELECTRICAL HAZARD. Plug the system into a properly
grounded receptacle with adequate current capacity.

Chemical Safety

Read and comprehend all safety data sheets (SDSs) by chemical manufacturers
before you use, store, or handle any chemicals or hazardous materials.

Wear personal protective equipment (gloves, safety glasses, fully enclosed shoes,
lab coats) when handling chemicals.

Do not inhale fumes from chemicals. Use adequate ventilation, and return caps to
bottles immediately after use.

Appendix B: Safety

Electrical Safety

Check regularly for chemical spills or leaks. Follow SDS recommendations for
cleaning up spills or leaks.

Disposal of Products

Used IFCs should be handled and disposed of in accordance with federal, state,
regional, and local laws for hazardous waste management and disposal.

Do not dispose of this product in unsorted municipal waste. This equipment may
contain hazardous substances that could affect health and the environment. Use
appropriate take-back systems when disposing of materials and equipment.

Learn more at fluidigm.com/compliance

Genotyping with Juno Getting Started Guide

31

Back Cover

7000 Shoreline Court, #100
South San Francisco, CA
T: 650 266 6000

For technical support visit
fluidigm.com/support.