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The world leader in serving science

Experimental Workflow on the QuantStudio™ 3D Digital PCR

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Absolute

Concentration measurements are output in

target copies/μL

Simple

Turn-key chip-based workflow – load & go

QuantStudio™ 3D is based on a simple and affordable platform with

enough data points to satisfy many of current applications

QuantStudio™ 3D – Digital PCR For Everyone

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Instrument reads one chip at a time

Less than a minute to read one chip

Factory calibrated to detect FAM™,

SYBR Green, VIC® and ROX® dyes

Intuitive touch screen operation

Small footprint

• 22.5cm x 13.5cm x 23cm

• No additional laptop or computer

QuantStudio™ 3D Digital PCR System

Automatic upload of results to cloud for analysis

Instrument stores last 100 chips results

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20,000 reaction wells per chip

Minimal sample loss

One sample per chip

Simple and consistent loading

Sealed consumable minimizes

contamination

Each chip identified by unique

identification number

QuantStudio™ 3D Digital PCR 20K Chip

Fixed reaction volume minimizes upfront sample

manipulation

20,000 Reaction-wells

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Preparing the DNA Samples

•DNA Quality

• Use a optimized DNA extraction protocol

• salting-out procedures and crude lysates are not recommended

• Make sure DNA extracts do not contain PCR inhibitors

• A260/230 and A260/280 ratios should be between 1.7 and 1.9

• ~2.0 for RNA

• Make sure DNA is not degraded

• E.g. as visualized on an agarose gel

• Not heated >60°C as this may cause degradation

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Preparing the DNA Samples, continued

•DNA Quantity

• The quantity of sample added to a digital PCR reaction depends on the

• Concentration of genomic or complementary DNA (gDNA or cDNA) present in each sample

• Number of copies of the target sequence present in the genome or total RNA of your samples

•DNA Quantity

• We recommend the following methods of quantitation:

• Quant-iT™ assay nucleic acid quantitation using the Qubit® Quantitation Platform (fluorimetry)

• Spectrophotometry (OD260)

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Sample dilution

•Samples are diluted down to a limiting quantity, such that most individual PCR reactions contain either zero or one target molecule

•Two scenarios:

• Determine the optimal dilution when the target copy number per genome is known

• Determine the optimal dilution when the target copy number per genome is unknown

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Sample dilution when the target is known

•Goal is to dilute the samples so that each partition will contain approximately 0.6 to 1.6 copies of the target sequence

•Example

• Human genomic DNA has 3.3 pg/copy of a given gene

• Each partition is 865 pL

• To obtain 0.6 copies/partition

• 0.6 copies / 809 pL approximately 741 copies/µL or 2.45 ng/µL

• Assuming 3.3 pg/copy of a given gene in the human genome, and a reaction-well volume of 755 pL (v2 chip)

• 2. Dilute the stock gDNA in a given sample to ~1045 copies/μL or 3.45 ng/μL in the final reaction, resulting in ~0.73 copies per reaction well.

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When Target Copy Number is Unknown

•This will be the case when

• The number of copies of a specific sequence per genome is unknown

• The expression level of the RNA of interest is unknown

•The optimal dilution is determined by loading and imaging a dilution series of the sample at the expected digital range

•To capture the digital range, assay three to four data points above and below the expected digital range

• If there is real-time data from a qPCR experiment available, this can be a guide for the starting and end points of the dilution series

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Prepare the digital PCR reactions

•Required items

• TaqMan® Assay(s)

• QuantStudio™ 3D Digital PCR Master Mix

• Pipettes and tips, P10 to P1000

• Reaction tubes

• Molecular grade water

• Microcentrifuge

• Vortexer

• Gloves, marker pen, lint-free wipes

Material Volume

(μL)

Stoc

k

Final

QuantStudio™

3D Digital PCR

Master Mix, 2X

7.25 2X 1X

TaqMan®

Assay, 20X

(primer/probe

mix)

0.725 20X 1X

Diluted DNA 1.5 23

ng/µL*

2.3

ng/µL*

Water 5.025 - -

Total volume

( sample/1 chip) 14.5 - - * Just an example, it will

depend on the application

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Required Instrumentation

QuantStudio™ 3D Digital PCR

Chip Loader (PN 4482592)

GeneAmp® PCR System

9700 (PN 4428234)

QuantStudio™ 3D Digital

PCR Instrument (PN 4482593)

QuantStudio™ 3D Tilt Base

For GeneAmp® PCR System

9700 (PN 4486414)

QuantStudio™ 3D Adapter

Kit For GeneAmp® PCR

System 9700 (PN 4485513)

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QuantStudio™ 3D Workflow with Chip Loader

Chip & rxn mix

equilibrate to

room temp (~15’)

Apply lid

Inject

immersion

fluid

Seal chip in

UV sealing

station

Cycle chip on GeneAmp®

PCR System 9700

Read chip on

QuantStudio™ 3D

Digital PCR Instrument

Place chip and lid on

loader & load 14.5 μL of

rxn mix

several drops

of immersion

fluid

11° Front

Tilt

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Loading Reactions

• Required items

• Prepared samples/dilutions

• QuantStudio™ 3D Digital PCR 20K Chips

• QuantStudio™ 3D Digital PCR Chip Case Lids

• QuantStudio™ 3D Digital PCR Sample Loading Blades

• QuantStudio™ 12K Flex OpenArray® Immersion Fluid

• QuantStudio™ 12K Flex OpenArray® Immersion Fluid Tip

• Isopropanol, 10% solution (or ethanol)

• Molecular grade water

• Microcentrifuge, vortex, pipettes

• Gloves, scissors, lit-free wipes

Note! Never touch the inside of the Chip. If so,

DISCARD IT.

IMPORTANT! Never handle Chips or Chip Case

Lids without gloves.

Oils from your hands can contaminate the

components and interfere with thermal cycling

and imaging.

Note! Do not touch the inside of the Lid. If so,

CLEAN it with a isopropanol or ethanol sprayed

wipe

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Material Prep

2. Prepare the syringe containing

Chip Sealant

• Place the assembled syringe within

its protective package until ready

for use.

1. Prepare a syringe containing

Immersion Fluid:

a) Before uncapping the syringe,

gently pull back the plunger 1-

2 mm and release it to break

any resistance that may have

formed during storage.

b) Unscrew the cap from the

syringe, then attach the Tip by

pushing it into place.

3. Prepare the Loading Blade,

the Lid and the Chip, removing

them form their packages

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Loading Reactions

• Open Chip package, gently grasp the chip by its sides, then place it

face-up on the heated block, preheated to 46°C.

• IMPORTANT! Allow the Chip to sit on the heated block for at least 5

seconds before loading it.

• IMPORTANT! Place only one Chip on the heated block at a time.

• Note: If desired, use a permanent pen or marker to label the back of Chip.

Marking the back of the chips will not affect the imaging data.

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Inspect the chip for problems

•Visually inspect the sealed Digital PCR 20K Chip for potential problems:

• Leaks – Confirm that no Immersion Fluid is leaking from the fill port or around the seal between the Chip Case and lid.

• Bubbles – Confirm that the Chip Case is free of excessive bubbles. One small air bubble is acceptable.

• Correct lid orientation – Confirm that the Chip Case Lid and the Digital PCR 20K Chip are correctly aligned.

Thermal cycle the Chips within 2 hours

after loading them.

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QuantStudio™ 3D Workflow

50’ 2hr 25’

Read Load Thermal Cycle

24 Samples

3hrs 6hrs 9hrs 0hrs

Lunc

h

Optimized Run Schedule Enables 96 Samples Per Day

Read can be

completed

next day

2’ per chip 1’ per chip

3 hrs 15’

Run Time

24 chips Run 1

Run 2 24 chips Run 2

Run 3 24 chips Run 4

Run 4 24 chips Run 4

7 hrs 20 min 9 hrs 15 min

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Thermal Protocol

Step Temp (°C) Time (mm:ss)

Hold Polymerase activation 96.0 10:00

Cycling

(39 cycles)

Anneal/extend 60.0 2:00

Denature 98.0 00:30

Hold Final extension 60.0 2:00

Hold (optional) Storage 10.0 99:59

• Program the GeneAmp® 9700 system • Any protocol optimization would be done by changing the

anneal/extension temperature • Reaction Volume: 20 l • Ramp Mode: Flat

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Running the chips on the 9700

• Confirm that the Tilt Base for Dual Flat Block 9700 System is installed beneath the thermal cycler

• Load the chips in the Chip Carrier

• The Chip Carrier has a set of four alignment pegs that fit into the holes of a flat sample block

• Lay the Thermal Pads over the Digital PCR 20K Chips

• Slide the heated cover forward and pull the heated cover lever down to engage the cover with the Chips

• Position the 24 chips so the fill port is oriented toward the front of the GeneAmp® PCR System 9700 and bubbles go to the top

• Start the PCR run

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Loading less than 24 Chips

• If you are thermal cycling less than 24 Digital PCR 20K

Chips, load according to the following recomendations:

• Load each Digital PCR 20K Chip so that the fill port is oriented toward

the front of the GeneAmp® PCR System 9700.

• Place at least 1 chip on the right sample block.

• Balance the load between the left and right sample blocks.

• Load the chips to the center positions of the sample block first, then

work outwards as shown below:

• For example, if thermal cycling 4 chips, load them to positions 1 through 4.

If you are thermal cycling 7 chips, load them to positions 1 through 6 and to

either position 7 or 8).

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Before reading the chips

• Visually inspect each Digital PCR 20K Chip for signs of leaks or potential problems.

• Using a lint-free laboratory wipe, remove any condensation or Immersion Fluid from the surface of the chip by wiping in one direction

• Run the prepared Digital PCR 20K Chips within 1 hour after thermal cycling them if left at room temperature.

• After thermal cycling, the Digital PCR 20K Chips can remain on the sample block for up to 24 hours.

Use 10% isopropanol or

ethanol solution to remove

any

dried residue

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Reading the chips

• Set the destination for the imaging data

• You may for example use a USB memory stick to collect the run data

• Open the chip tray and load the Digital PCR 20K Chip face-up into the bay. Confirm that the Digital PCR 20K Chip is correctly aligned within the chip tray, then close it.

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Reading the chips, continued

• Touch Start Run, then wait for the QuantStudio™ 3D Instrument to image the chip

• While the instrument images the chip, the touch screen counts down the time remaining in the run

• It will take less than 30 seconds to read the chip

• When the touch screen displays the Analyzing Chip screen, open the chip tray and remove the Digital PCR 20K Chip

• Store or discard it as desired.

• You can read another Digital PCR 20K Chip without waiting for the analysis to complete

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Reviewing the Results

• FAM™ and VIC® data will be shown as the instrument has three filters:

• FAM ™

• VIC®

• ROX ®

• After reviewing the results of the run, touch Done

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After a Digital PCR 20K Chip has been read

• You run the imaged Digital PCR 20K Chip again for up to 1 hour after thermal cycling.

• If you read multiple Chips in rapid succession, touch the scroll buttons to review the results of the previously imaged chips.

• The Instrument retains a copy of the analyzed data for the imaged Chip that you can access from the Run History screen.

Note: The results will remain in

the QuantStudio™ 3D Instrument

cache for approximately 100

readings. After 100 chips, the

instrument removes the oldest

data file in the cache to store each

new reading

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Questions

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The QuantStudio™ 3D Digital PCR System is For Research

Use Only. Not for use in diagnostic procedures.

© 2014 Thermo Fisher Scientific Inc. All rights reserved. All

trademarks are the property of Thermo Fisher Scientific and

its subsidiaries unless otherwise specified. TaqMan is a

trademark of Roche Molecular Systems, Inc., used under

permission and license.