Thermo Fisher Scientific Oncomine Focus Assay, Part IV: Sequencing User guide

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

Oncomine™ Focus Assay, Part IV: Sequencing

USER GUIDE

for use with:

Ion PGM™ Select Sequencing Kit

Catalog Number A28396

Publication Number MAN0013532

Revision C.0

The information in this guide is subject to change without notice.

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history

Revision history of MAN0013532

Revision Date Description

C.0 8 February 2016 Updated the Oncomine™ Focus Assay documentation set with information about Torrent

Suite™ Assay Development Software

B.0 July 22, 2015 •Clariﬁed and edited ISP pellet handling and chip loading steps

• Corrected minor typos

A.0 May 22, 2015 • New guide for new product

Important Licensing Information

These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

applicable Limited Use Label Licenses.

Corporate entity

Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS

All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed.

Contents

■CHAPTER 1 Product information ....................................... 6

Product description ............................................................. 6

Template kit compatibility ........................................................ 6

System and software compatibility ................................................ 6

Ion PGM™ Dx System instructions ................................................. 7

Materials provided .............................................................. 7

Instrument systems ............................................................. 8

Required materials and equipment (not provided) ................................... 8

Ion PGM™ Sequencer ............................................................ 9

■CHAPTER 2 Before you begin .......................................... 11

Precautions before using the Ion PGM™ System .................................... 11

Update the software ........................................................ 11

Instrument installation by trained personnel only .............................. 11

CO2 contamination ......................................................... 11

Instrument vibration and clearances ......................................... 11

Static electricity ........................................................... 11

Ventilation requirements ................................................... 12

Procedural guidelines .......................................................... 12

Chip handling and use guidelines ................................................ 12

Gas cylinders .................................................................. 13

Perform a leak test ........................................................ 13

Protocol workﬂow .............................................................. 14

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

3

■CHAPTER 3 Clean and initialize ....................................... 15

Ion PGM™ Dx System users ...................................................... 15

Clean the instrument ........................................................... 15

Materials required ......................................................... 15

Cleaning schedule ......................................................... 15

Cleaning setup ............................................................ 16

18 MΩ water cleaning ...................................................... 16

Chlorite cleaning .......................................................... 17

Initialize the Ion PGM™ System ................................................... 18

Materials required ......................................................... 18

Initialization guidelines ..................................................... 19

Before initialization ........................................................ 19

Prepare SEQ Wash 2 Bottle ................................................. 19

Prepare the SEQ Wash 1 and SEQ Wash 3 bottles .............................. 20

Begin initialization ......................................................... 21

Prepare the 50‑mL reagent bottles with SEQ dNTP solutions .................... 22

Attach the sipper tubes and Reagent bottles .................................. 22

■CHAPTER 4 Sequencing protocol ..................................... 24

Ion PGM™ Dx System users ...................................................... 24

Materials required ............................................................. 24

Before starting ................................................................ 25

Anneal the SEQ Primer ......................................................... 25

Set up the sequencing run ....................................................... 27

Bind SEQ Enzyme .............................................................. 29

Prepare and load chip .......................................................... 29

Remove liquid from the chip ..................................................... 29

Load the chip .................................................................. 31

Select the Planned Run and perform the run ...................................... 33

Select the Planned Run ..................................................... 33

Perform the run ........................................................... 34

■APPENDIX A Troubleshooting ......................................... 35

Chip Check .................................................................... 35

Chip calibration (before loading sample) .......................................... 36

Chip calibration (after loading sample) ............................................ 38

Initialization—General errors .................................................... 39

Initialization—Auto pH errors .................................................... 40

Initialization—Reagent pH veriﬁcation ............................................ 45

Contents

4

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

■APPENDIX B Manually adjust W2 pH .................................. 47

Materials and equipment needed ................................................ 47

Procedure ..................................................................... 47

■APPENDIX C Sequencing run times ................................... 49

■APPENDIX D Additional instrument information .................... 50

Ion PGM™ Sequencer input and output connections ................................. 50

Power the Ion PGM™ Sequencer on or off .......................................... 51

Power on ................................................................. 51

Power off ................................................................. 51

Update the Ion PGM™ System software ............................................ 51

■APPENDIX E Safety ..................................................... 52

Symbols on this instrument ..................................................... 52

Safety alerts on this instrument .................................................. 53

Safety information for instruments not manufactured by Thermo Fisher Scientiﬁc ..... 54

Instrument safety .............................................................. 54

General .................................................................. 54

Physical injury ............................................................ 54

Electrical ................................................................. 54

Cleaning and decontamination ............................................... 55

Laser .................................................................... 55

Safety and electromagnetic compatibility (EMC) standards .......................... 55

Safety .................................................................... 55

EMC ...................................................................... 55

Environmental design ...................................................... 56

Chemical safety ................................................................ 56

■Documentation and support ............................................. 57

Customer and technical support ................................................. 57

Limited product warranty ....................................................... 57

Contents

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

5

Product information

Product description

The Ion PGM™ Select Sequencing Kit (Cat. no. A28396) includes reagents and

materials for sequencing up to ~200-bp inserts using the Ion 318™ Select Chip. It also

includes components for cleaning and initializing the sequencer.

The kit includes reagents and materials for four sequencer initializations.

Sequencing type Number of ﬂows

(cycles)

Number of runs per

kit

Maximum number

of runs per

initialization

200-base-read 500 (125) 8 2

Template kit compatibility

For optimal performance, we recommend the Ion OneTouch™ Select Template Kit.

System and software compatibility

The kits and components described in this guide are compatible with the following

instrument systems and software:

• The Ion PGM™ System with Torrent Suite™ Software version 5.0 or later. To

update the system software, see the Oncomine™ Focus Assay, Part IV: Sequencing

User Guide (Pub. No. MAN0013532).

• The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software

version 5.0 or later, for Research Use Only experiments only.[1]

1

[1] The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software is For Research

Use Only. Not for use in diagnostic procedures.

6

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

Ion PGM™ Dx System instructions

If you are using components of the Ion PGM™ Select Sequencing Kit on the Ion PGM™

Dx Sequencer with Torrent Suite™ Assay Development Software (For Research Use

Only experiments), follow the instrument instructions provided in the Ion PGM™ Dx

System, Part IV: Sequencing User Guide (Pub. No. 4474247).

The Ion PGM™ Dx Sequencer workow and steps dier from those described in this

user guide.

Materials provided

The Ion PGM™ Select Sequencing Kit (Cat. no. A28396) includes the following

modules and components, and is also included as a part of the Oncomine™ Focus

Assay Package Solution (Cat. no. A28548).

✓Component Color Amount Storage

Ion PGM™ Select Sequencing Supplies (Part no. A28393)

SEQ Wash Bottle Sippers Gray 8 long, 16 short 15°C to 30°C

SEQ Reagent Tube Sippers Blue 32

50-mL Centrifuge Tubes — 2 × 25

SEQ Wash 1 Bottle — 1

SEQ Wash 2 Bottle — 1

SEQ Wash 3 Bottle — 1

SEQ Reagent Tube Labels — 1 set

Ion PGM™ Select Sequencing Reagents (Part no. A28391)

SEQ dGTP Black 2 × 40 µL –30°C to –10°C

SEQ dCTP Blue 2 × 40 µL

SEQ dATP Green 2 × 40 µL

SEQ dTTP Red 2 × 40 µL

SEQ Enzyme Yellow 24 µL

SEQ Primer White 96 µL

Ion PGM™ Select Sequencing Solutions (Part no. A28392)

SEQ W2 Solution White 8 × 126.25 mL 2°C to 8°C[1]

SEQ Cleaning Tablet N/A 8 tablets

SEQ Sample Buffer Brown 1 × 160 µL

SEQ W3 Solution White 4 × 100 mL

Chapter 1 Product information

Ion PGM™ Dx System instructions

1

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

7

[1] Store W2 Solution protected from light.

✓Component Amount Storage

Ion 318™ Select Chips 8 15°C to 30°C

Instrument systems

This sequencing kit is designed for use with the following instrument systems,

ordered separately.

Components Catalog no.

Ion PGM™ System

Ion PGM™ System and accessories 4462921

Ion PGM™ Torrent Server 4462918

Ion PGM™ Chip Minifuge:

120 VAC

230 VAC

4479672

4479673

Ion PGM™ Dx System

Ion PGM™ Dx System bundle A25511

Required materials and equipment (not provided)

Unless otherwise indicated, all materials are available through thermosher.com.

MLS: Fisher Scientic (www.sherscientic.com) or other major laboratory supplier.

3Item Source Quantity

One of the following:

• Veriti™ Dx 96-Well Thermal Cycler, 0.2 mL

• GeneAmp™ PCR System 9700 or Dual 96-well

Thermal Cycler

• AB™ 2720 Thermal Cycler

• ProFlex™ 96-Well PCR System

See web product

pages

1

Tank of compressed nitrogen (grade 4.8, 99.998% or

better)

MLS 1

Multistage (dual-stage) gas regulator (0-50 PSI, 2-3

Bar output)

VWR

International,

55850-422

1

18-MΩ water puriﬁcation system MLS 1

Chapter 1 Product information

Instrument systems

1

8

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

3Item Source Quantity

0.45 ‑µm vacuum ﬁltration system and ﬁlters (nylon

or PVDF ﬁlters, 1 L volume)

MLS 1

Pipettors (2-, 20-, 200-, and 1000-µL) MLS 1 each

Low-retention aerosol-barrier pipette tips (10-, 20-,

200-, and 1000-µL)

MLS 10

racks/size

Vortexer with a rubber platform MLS 1

Picofuge MLS 1

Microcentrifuge with adapter for 0.2-mL tube MLS 1

Ion OneTouch™ 2 Rack Kit A24694 1

Graduated cylinders (1 L or 2 L volume) MLS 1

Glass bottle (1 L) MLS 1

15-mL conical tubes MLS 1

NaOH, ACS grade (10 M) MLS —

Nuclease-Free Water MLS —

Ion PGM™ Sequencer

The following image depicts the major components of the Ion PGM™ Sequencer. The

Ion PGM™ Dx Sequencer has the same components.

E

B

C

D

A

H

G

F

I

Chapter 1 Product information

Ion PGM™ Sequencer

1

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

9

Label Component

A Touchscreen

B Chip clamp

C Grounding plate

D Power button

E Reagent bottles

F Wash 1 Bottle (W1 position)

G Wash 2 Bottle (W2 position)

H Wash 3 Bottle (W3 position)

I Waste Bottle

Chapter 1 Product information

Ion PGM™ Sequencer

1

10

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

Before you begin

Precautions before using the Ion PGM™ System

For additional safety information, see Appendix E, “Safety“.

IMPORTANT! Before proceeding, make sure that you have updated the Torrent

Suite™ and Ion PGM™ System software to Version 5.0 or later. See “Update the Ion

PGM™ System software“ on page 51.

IMPORTANT! The Ion PGM™ System is installed by trained service personnel and

must not be relocated without assistance from trained service personnel. See

“Customer and technical support“ on page 57.

IMPORTANT! Dry ice (solid CO2) must be kept away from areas where buers, wash

solutions, or sources of molecular biology grade water for the Ion PGM™ System are

used. High air concentrations of subliming CO2 may change the pH of such buers

during or after their preparation. The stability of the pH of these buers is a critical

factor in the performance of the Ion PGM™ System.

IMPORTANT! Signicant vibration during sequencing may add noise and reduce the

quality of the measurements. The Ion PGM™ System must be installed on a bench that

is free from vibrations or in contact with equipment that can cause vibrations to the

bench (freezers, pumps, and other similar equipment).

IMPORTANT! Position the Ion PGM™ System so that the front bezel is a minimum of

12 in. (30.5 cm) and the Reagent Tubes containing dNTPs are a minimum of 8 in.

(20.3 cm) from the front of the laboratory bench. Place the instrument at least 40 in.

(1 meter) away from major sources of electronic noise such as refrigerators or

microwaves.

IMPORTANT! To avoid possible damage to the chip from static electricity, you must

ground yourself on the grounding plate (located next to the chip clamp) prior to

handling chips, and do not place chips on non-grounded surfaces such as a bench.

Always use the grounding plate to hold chips that are not in the package or inserted

in the chip clamp or the Ion PGM™ Chip Minifuge bucket.

2

Update the

software

Instrument

installation by

trained personnel

only

CO2 contamination

Instrument

vibration and

clearances

Static electricity

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

11

WARNING! Instrumentation must be installed and operated in a well-

ventilated environment as dened as having a minimum airow of 6–10 air

changes per hour. Assess the need for ventilation or atmospheric monitoring to

avoid asphyxiation accidents from inert gases and/or oxygen depletion, and

take measures to clearly identify potentially hazardous areas through training

or signage. Please contact your Environmental Health and Safety Coordinator

to conrm that the instruments will be installed and operated in an

environment with sucient ventilation.

Procedural guidelines

•Use good laboratory practices to minimize cross-contamination of products.

When designing the laboratory layout, consider the need for space separation of

pre- and post-amplication activities. Dedicate laboratory supplies and/or

equipment to the appropriate space to signicantly reduce the potential for

contamination.

•Pipet viscous solutions slowly and ensure complete mixing.

• Ensure all reagents are completely thawed at room temperature, i.e., no ice

crystals are visible.

• Vortex all reagents, except for enzymes, for 5 seconds (enzymes should be mixed

by icking the tube with your nger four times). Pulse spin for 3-5 seconds before

use.

• When performing two sequencing runs from the same initialization, the second

run must be started within 27 hours after initialization.

• If you press the Abort buon on the touchscreen during any procedure, the

touchscreen may freeze and you may need to restart the sequencer.

Chip handling and use guidelines

• Always remove gloves before transferring chips on or o the instrument. Hold

chips by their edges when handling.

• To avoid damage due to electrostatic discharge (ESD), do not place chips directly

on the bench or any other surface. Always place chips either on the grounding

plate on the Ion PGM™ Sequencer or in the Ion PGM™ Chip Minifuge bucket.

• Used chips cannot be reused for sequencing. Used chips may be marked for

cleaning and initialization.

Ventilation

requirements

Chapter 2 Before you begin

Procedural guidelines

2

12

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

Gas cylinders

You must supply the required nitrogen gas cylinder and accessories for the

installation. This instrument requires a pressurized house line, or one size 1-A

nitrogen gas cylinder that holds approximately 7.2 m3 (257 ft3) of gas when full. Use

only prepuried nitrogen of 99.995% (grade 4.5) or greater purity.

CAUTION! Damage to the instrument and its products can result from using

impure gas, gases other than nitrogen, or an inadequate amount of gas.

WARNING! EXPLOSION HAZARD. Pressurized gas cylinders are potentially

explosive. Always cap the gas cylinder when it is not in use, and aach it rmly

to the wall or gas cylinder cart with approved brackets or chains.

WARNING! Gas cylinders are heavy and may topple over, potentially causing

personal injury and tank damage. Cylinders should be rmly secured to a wall

or work surface. Please contact your environmental health and safety

coordinator for guidance on the proper installation of a gas cylinder.

To perform a leak test on the gas cylinder:

1. Open the main tank shuto valve. The gas tank regulator’s high-pressure gauge

should read approximately 2000–2500 psi for a full tank.

2. Adjust the pressure to the instrument by slowly turning the pressure adjustment

valve clockwise until the low-pressure gauge reads 30 psi.

3. Close the needle valve, then close the main tank valve.

4. Monitor the high-pressure gas tank regulator gauge for 5 minutes. There should

be no noticeable drop in pressure.

If the pressure... Then...

Drops in 5 minutes There may be a leak at either the needle valve or the gas tank

regulator itself. Check the ﬁttings and resolve any problems,

then continue with the following steps.

Does not drop in

5 minutes

The instrument passes the leak test. Re-open the main tank

valve and skip the following steps.

5. Open the main tank valve and the needle valve for at least 15 seconds to

pressurize the instrument.

6. Close the main tank valve.

7. Monitor the high-pressure gas tank regulator gauge. There should be no more

than a 100-psi drop in pressure after 5 minutes. Locate and resolve any leaks.

Turn the main tank valve back on.

Perform a leak

test

Chapter 2 Before you begin

Gas cylinders

2

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

13

Protocol workﬂow

Note: Up to two sequencing runs may be performed from one initialization, if both

runs are started within 27 hours after initialization.

“Clean the instrument“ on page 15

q

“Initialize the Ion PGM™ System“ on page 18

q

“Load the chip“ on page 31

q

“Perform the run“ on page 34

Chapter 2 Before you begin

Protocol workﬂow

2

14

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

Clean and initialize

Ion PGM™ Dx System users

IMPORTANT! If you are using the Ion PGM™ Dx System with Torrent Suite™ Assay

Development Software (For Research Use Only experiments), follow the instrument

instructions provided in the Ion PGM™ Dx System, Part IV: Sequencing User Guide (Pub.

No. 4474247).[2]

The Ion PGM™ Dx Sequencer workow and steps dier from those described in this

user guide.

Clean the instrument

•18 MΩ water (e.g., the ELGA™ PURELAB™ Flex Water Purication System)

•Cleaning boles and collection trays (provided with the Ion PGM™ System)

• Old chip that has been used for sequencing, marked for cleaning

Note: Always keep a used chip in the instrument when it is not in use. This

prevents air from entering the system.

• Used sipper tubes (from the previous run)

• Squirt bole

•Chlorite cleaning: SEQ Cleaning Tablet (provided in the kit)

•Chlorite cleaning: 1 M NaOH, diluted fresh each week from 10 M NaOH

•Chlorite cleaning: Glass bole (1 L)

•Chlorite cleaning: 0.22-µm or 0.45-µm vacuum ltration system and lters

The Ion PGM™ Sequencer requires cleaning with either 18 MΩ water or a chlorite

solution every time the instrument is initialized.

Clean with… Schedule

18 MΩ water • Daily, when instrument is in use (e.g., not necessary on weekends)

•After one or more runs totaling ≤1100 ﬂows

• If more than 27 hours but less than 48 hours have elapsed between

the last cleaning/initialization and the start of a run

• If you cleaned with chlorite a week ago and have not used the

instrument since then

3

Materials required

Cleaning schedule

[2] The Ion PGM™ Dx System with Torrent Suite™ Assay Development Software is For Research

Use Only. Not for use in diagnostic procedures.

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

15

Clean with… Schedule

Chlorite

solution

• Once a week, unless the instrument has not been used since the last

chlorite cleaning (in which case, clean with 18 MΩ water before using)

• If the instrument has been left with reagents for more than 48 hours

(for example, over the weekend)

IMPORTANT! For all the following steps, use 18 MΩ water directly from the

purication system. Do not use water that has been collected or stored in any other

containers.

• Remove any wash and reagent boles that are aached to the Ion PGM™ System

before cleaning.

•Do not remove old sippers before cleaning. The sippers are used as part of the

cleaning procedure.

• Old chips that have been used for sequencing can be marked and used in the

cleaning procedure:

– Write "CL" on the corner of used chips designated for chlorite washes only

(do not use these chips for initialization)

– Write "W" on the corner of used chips for water washes and initialization (do

not use these chips for chlorite washes)

• Wash boles (250 mL and 2 L) provided as part of instrument installation can be

marked and used for cleaning. After you have used the wash boles provided

with the sequencing kit for the specied number of runs, you can use them as

extra cleaning boles. Mark them for cleaning use only.

1. Empty any remaining solution from each cleaning bole (two 250-mL boles and

one 2-L bole) and rinse each bole twice with ~100 mL of 18 MΩ water.

2. Press Clean on the touchscreen, and select the 18-MOhm water cleaning

checkbox. Press Next.

3. Using ungloved hands, secure a used chip designated for cleaning in the chip

clamp.

IMPORTANT! Always make sure that both red rubber gasket port ings are

securely in place when securing chips with the chip clamp. Failure to do so can

result in a spill hazard and instrument damage.

4. Remove all wash and reagent boles aached to the instrument. Keep the sippers

in place at all positions. Press Next.

5. Add 250 mL of 18 MΩ water to an empty 250-mL cleaning bole.

6. Rinse the outside of the sipper tube in the W1 position on the instrument with a

squirt bole containing 18 MΩ water.

7. Aach the 250-mL bole containing 18 MΩ water to the W1 position, ensuring

that the W1 cap is screwed on tightly. Press Next.

Cleaning setup

18 MΩ water

cleaning

Chapter 3 Clean and initialize

Clean the instrument

3

16

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

8. Place the empty 2-L cleaning bole in the W2 position and the empty 250-mL

bole in the W3 position, and insert the sippers into the boles. Do not screw on

the caps.

9. Place collection trays below the reagent sippers in the dNTP positions. Press

Next to begin cleaning.

10. When cleaning is complete, remove the boles and sippers from the W1, W2 and

W3 positions. Leave the reagent sippers and collection trays in place. Press Next

to return to the main menu and proceed to initialization.

Note: Prepare a stock of 1 M NaOH each week by diluting 10 M NaOH with 18 MΩ

water.

1. Empty any remaining solution from each cleaning bole (two 250-mL boles and

one 2-L bole) and rinse each bole twice with ~100 mL of 18 MΩ water.

2. Fill a glass bole with 1 L of 18 MΩ water and add an SEQ Cleaning Tablet

(chlorite tablet). Allow the tablet to completely dissolve (~10 minutes).

3. When the tablet has dissolved, add 1 mL of 1 M NaOH.

4. Mix by inverting the bole 10 times and lter the solution using a 0.22-µm or

0.45-µm lter. Use the chlorite solution within 2–3 hours. Discard any unused

solution after this time.

5. Press Clean on the touchscreen, and select the Chlorite cleaning checkbox. Press

Next.

6. Using ungloved hands, secure a used chip designated for cleaning in the chip

clamp.

IMPORTANT! Always make sure that both red rubber gasket port ings are

securely in place when securing chips with the chip clamp. Failure to do so can

result in a spill hazard and instrument damage.

7. Remove all wash and reagent boles aached to the instrument. Keep the sippers

in place at all positions. Press Next.

8. Add 250 mL of the ltered chlorite solution to an empty 250-mL cleaning bole.

9. Rinse the outside of the sipper tube in the W1 position on the instrument with a

squirt bole containing 18 MΩ water.

10. Aach the 250-mL bole with the ltered chlorite solution to the W1 position.

Make sure that the W1 cap is tight. Press Next.

11. Place the empty 2-L cleaning bole in the W2 position and the empty 250-mL

bole in the W3 position, and insert the sippers into the boles. Do not screw on

the caps.

12. Place collection trays below the reagent sippers in the dNTP positions. Press

Next to begin cleaning.

Chlorite cleaning

Chapter 3 Clean and initialize

Clean the instrument

3

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

17

13. When prompted, remove the bole containing the chlorite solution from the W1

position.

14. Rinse the outside of the W1 sipper tube with a squirt bole containing 18 MΩ

water.

15. Fill a clean 250-mL bole with 250 mL of 18 MΩ water and aach the bole in the

W1 position. Make sure the cap is tight. Press Next to begin the water rinse.

16. When cleaning is complete, remove the boles and sippers from the W1, W2 and

W3 positions. Leave the reagent sippers and collection trays in place.

17. Press Next to return to the main menu and proceed to initialization.

Initialize the Ion PGM™ System

Initialization takes ~1 hour. As part of the initialization process, rst prepare the Wash

and Reagent Boles as described in this section.

Materials provided in the kit

•Wash 1, 2, and 3 Boles and sipper tubes

• Reagent Boles and sipper tubes

• SEQ dGTP

• SEQ dCTP

• SEQ dATP

• SEQ dTTP

• SEQ W2 Solution (stored protected from light)

• SEQ W3 Solution

Other materials and equipment

• Used chip (leave chip on the instrument during initialization)

• 18 MΩ water

• 100 mM NaOH (prepared daily)

• Ice

• Filtered and unltered pipee tips and pipees

• Vortex mixer

• Microcentrifuge

Materials required

Chapter 3 Clean and initialize

Initialize the Ion PGM™ System

3

18

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

IMPORTANT! Handle nucleotides carefully to avoid cross-contamination. Always

change gloves after removing used sipper tubes from the Ion PGM™ System to avoid

cross contamination of the nucleotides. Also change gloves after handling

concentrated dNTP stocks.

For each initialization, the rst run should be started within 1 hour after initialization,

and the last run must be started within 27 hours after initialization.

1. Remove the dNTP stock solutions from the freezer and thaw on ice.

2. Check the tank pressure for the nitrogen gas. If the tank pressure is below

500 psi, change the tank.

3. Note the mold line on the Wash 2 bole. If there are two mold lines on the bole,

mark the lower line to indicate that it is the correct one.

IMPORTANT! For all the following steps, pour the 18 MΩ water directly from the

purication system into the SEQ Wash 2 Bole. Do not use water that has been

collected or measured in any other containers.

1. Rinse the SEQ Wash 2 Bole (2 L) three times with 200 mL of 18 MΩ water.

2. If your 18 MΩ water system has a spigot, extend the water spigot into but not

below the neck of the SEQ Wash 2 Bole.

3. Fill the bole to the mold line. Volume of water will be ~2 liters.

Initialization

guidelines

Before

initialization

Prepare SEQ Wash

2 Bottle

Chapter 3 Clean and initialize

Initialize the Ion PGM™ System

3

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

19

4. Add the entire bole of SEQ W2 Solution to the SEQ Wash 2 Bole.

Note: Keep the SEQ W2 Solution bole to scan the barcode during the

initialization procedure.

5. Add 70 µL of freshly prepared 100 mM NaOH solution (not 1 M NaOH) to the

SEQ Wash 2 Bole.

Note: Dierent sites may require adding dierent volumes of 100 mM NaOH.

Some sites, for example, may require doubling the volume to 140 µL. See “Error

message: Added too much W1 to W2“ on page 44 for information on

determining the volume of 100 mM NaOH to add.

6. Cap the bole and invert ve times to mix, and immediately proceed through the

rest of the initialization procedure.

IMPORTANT! Do not store the mixed SEQ Wash 2 Bole.

Note: For the following steps, label the SEQ Wash 1 and SEQ Wash 3 Boles to avoid

confusion.

1. Rinse the SEQ Wash 1 and SEQ Wash 3 Boles three times with 50 mL of 18 MΩ

water.

2. SEQ Wash 1 Bole: Add 350 µL of freshly prepared 100 mM NaOH to the SEQ

Wash 1 Bole and cap the bole.

3. SEQ Wash 3 Bole: Add SEQ W3 Solution to the 50-mL line marked on the SEQ

Wash 3 Bole and cap the bole.

Prepare the SEQ

Wash 1 and SEQ

Wash 3 bottles

Chapter 3 Clean and initialize

Initialize the Ion PGM™ System

3

20

Oncomine™ Focus Assay, Part IV: Sequencing User Guide

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Thermo Fisher Scientific Oncomine Focus Assay, Part IV: Sequencing User guide

Thermo Fisher Scientific Oncomine Focus Assay, Part IV: Sequencing User guide

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