Thermo Fisher Scientific SOLiD™ EZ Bead™ Quick start guide

USER GUIDE

Applied Biosystems SOLiD® EZ Bead™ Enricher

Catalog Number 4448420

Publication Part Number 4443496 Rev. E

Revision Date October 2011

Emulsion Cleanup and

Bead Enrichment

Emulsion

PCR

Emulsion

Preparation

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Information in this document is subject to change without notice.

LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING

BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS

AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL,

INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT

LIMITED TO THE USE THEREOF.

NOTICE TO PURCHASER: LIMITED USE LABEL LICENSE: Research Use Only

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal

research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel.

This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and

commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional

rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries.

Covaris is a registered trademark of Covaris, Inc.

Latitude is a registered trademark of Dell.

Part Number 4443496 Rev. E

October 2011

Contents

3

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Safety alert words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Safety labels on instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■CHAPTER 1 Prepare and Run the SOLiD® EZ Bead™ Enricher . . . . . 11

Review the general workflow to prepare and sequence a library . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Materials and equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Prepare and install the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Run the Enricher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Run the Enricher using the control window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Concentrate and store the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Clean the Enricher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Prime 1: Prepare the Enricher before long-term storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Prime 2: Prepare the Enricher for first-time use or after long-term storage . . . . . . . . . . . . . 35

Modify the 3′ ends of the templated beads (E10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Extend the 3′ ends with Terminal Transferase and Bead Linker . . . . . . . . . . . . . . . . . . . . . . . . 39

Modify the 3′ ends of the templated beads (E20) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Extend the 3′ ends with Terminal Transferase and Bead Linker . . . . . . . . . . . . . . . . . . . . . . . . 41

Modify the 3′ ends of the templated beads (E80) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Extend the 3′ ends with Terminal Transferase and Bead Linker . . . . . . . . . . . . . . . . . . . . . . . . 42

Modify the 3′ ends of the templated beads (E120) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Extend the 3′ ends with Terminal Transferase and Bead Linker . . . . . . . . . . . . . . . . . . . . . . . . 43

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■APPENDIX A Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Required kits (E10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Required kits (E20) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Required kits (E80) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Required kits (E120) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Optional kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Contents

Required equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Optional equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Required consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

■APPENDIX B Supplementary Procedures . . . . . . . . . . . . . . . . . . . . . . . 59

Determine the template load per bead and the P2-positive percentage with the

SOLiD® Bead QC Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Prepare 10 µM SOLiD® Bead QC-Oligo-555 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Hybridize the samples and Calibrants with SOLiD® Bead QC-Oligo-555 . . . . . . . . . . . . . . . . . 61

Wash the beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Run calibrants on the flow cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Determine the template load: standard curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Determine the

P2-positive percentage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■APPENDIX C The Covaris® S2 System . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Operation notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Fill the tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Degas the water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Set the chiller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Perform required maintenance of the Covaris® S2 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Covaris® S2 programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Covalent

Declump 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Covalent

Declump 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

BeadHyb2X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

■APPENDIX D Instrument Warranty Information . . . . . . . . . . . . . . . . . . . 71

Computer configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Damages, claims, and returns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

■APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Instrumentation safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Symbols on instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Location of safety labels on instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

General instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Physical hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Electrical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

5

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Contents

Workstation safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

General chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Safety alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Specific alerts for chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

6Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Contents

7

Applied Biosystems SOLiD™ EZ Bead™ Enricher Getting Started Guide

About This Guide

Purpose

This getting started guide provides the information that you need to wash and enrich

templated beads on the SOLiD® EZ Bead™ Enricher.

To use the SOLiD® System, DNA libraries to be sequenced require amplification on

P1 Beads during emulsion PCR (ePCR), followed by enrichment of the templated

beads. Templated bead preparation can be performed using the SOLiD® EZ Bead

System:

•SOLiD

® EZ Bead Emulsifier: To prepare emulsions containing library template and

P1 Beads [refer to the Applied Biosystems SOLiD® EZ Bead Emulsifier Getting Started

Guide (Part no. 4441486)].

•SOLiD® EZ Bead Amplifier: To perform emulsion PCR (ePCR) [refer to the Applied

Biosystems SOLiD® EZ Bead Amplifier Getting Started Guide (Part no. 4443494)].

•SOLiD® EZ Bead Enricher: To wash and enrich the templated beads. After

enrichment, perform a Terminal Transferase reaction to modify the 3′ ends of the

library (see this guide).

The enriched beads are deposited on a slide and the library is sequenced (see Figure 1).

Figure1 The SOLiD EZ Bead System workflow

8Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

About This Guide

Prerequisites

This guide uses conventions and terminology that assume a working knowledge of the

Microsoft® Windows® operating system, the Internet, and Internet-based browsers.

Safety information

Note: For general safe alert on the instrument.

Safety alert words Four safety alert words appear in Life Technologies user documentation at points in

the document where you need to be aware of relevant hazards. Each alert word—

IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of

observation or action, as defined below:

IMPORTANT! – Indicates information that is necessary for proper instrument

operation, accurate chemistry kit use, or safe use of a chemical.

CAUTION! – Indicates a potentially hazardous situation that, if not avoided, may

result in minor or moderate injury. It may also be used to alert against unsafe practices.

WARNING! – Indicates a potentially hazardous situation that, if not avoided, could

result in death or serious injury.

DANGER! – Indicates an imminently hazardous situation that, if not avoided, will

result in death or serious injury. This signal word is to be limited to the most extreme

situations.

Note: Except for IMPORTANTs, each safety alert word in an Life Technologies

document appears with an open triangle figure that contains a hazard symbol. These

hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems

instruments (see “Safety symbols” on page 76).

SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by Applied Biosystems or

Ambion are available to you free 24 hours a day. For instructions on obtaining SDSs,

see “SDSs” on page 82.

IMPORTANT! For the SDSs of chemicals not distributed by Life Technologies or

Ambion contact the chemical manufacturer.

Safety labels on

instruments

The following CAUTION, WARNING, and DANGER statements may be displayed on

Life Technologies instruments in combination with the safety symbols described in the

preceding section.

9

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

About This Guide

Safety information

Hazard

symbol English Français

CAUTION! Hazardous chemicals. Read the Safety

Data Sheets (SDSs) before handling.

ATTENTION! Produits chimiques dangereux. Lire

fiche technique associée au produit avant toute

manipulation.

CAUTION! Hazardous waste. Refer to SDS(s) and

local regulations for handling and disposal.

ATTENTION! Déchets dangereux. Lire fiche

technique associée et prendre connaissance de la

régulation locale associées à la manipulation et

l'élimination des déchets.

10 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

About This Guide

Safety information

1

11

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Prepare and Run the SOLiD® EZ

Bead™ Enricher

This chapter explains how to prepare and run the SOLiD® EZ Bead™ Enricher:

“Review the general workflow to prepare and sequence a library” on page 12

“Before you begin” on page 13

“Prepare and install the reagents” on page 14

“Run the Enricher” on page 22

“Clean the Enricher” on page 27

Modify the 3’ ends of templated beads

(E10 on page 39, E20 on page 41, E80 on page 42, E120 on page 43)

12 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Review the general workflow to prepare and sequence a library

1

Review the general workflow to prepare and sequence a library

Materials and equipment required

See “Ordering Information” on page 47 for a list of equipment, kits, and consumables

necessary for this procedure.

Steps References

Quantitate/Titrate the library

5500 Series SOLiD® Systems Fragment Library

Preparation User Guide [Part no. 4460960)

5500 Series SOLiD® Systems Mate-Paired Library

Preparation User Guide [Part no. 4460958)

Emulsify the beads Applied Biosystems SOLiD® EZ Bead Emulsifier

Getting Started Guide (Part no. 4441486)

Amplify the beads Applied Biosystems SOLiD® EZ Bead Amplifier

Getting Started Guide (Part no. 4443494)

Enrich the templated beads

“Prepare and install the reagents” on page 14



“Run the Enricher” on page 22



“Clean the Enricher” on page 27

Modify the 3′ ends of the DNA E10 on page 39, E20 on page 41, E80 on page 42,

E120 on page 43

Deposit beads,

perform bead sequencing

5500 Series SOLiD® Sequencers Operation Guide

[Part no. 4456991)

13

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Before you begin 1

Before you begin

• Prepare an emulsion of the DNA library and P1 Beads with the SOLiD® EZ Bead

Emulsifier [refer to the Applied Biosystems SOLiD® EZ Bead Emulsifier Getting

Started Guide (Part no. 4441486)].

• Amplify the library template onto P1 beads using the SOLiD® EZ Bead Amplifier

[refer to the Applied Biosystems SOLiD® EZ Bead Amplifier Getting Started Guide

(Part no. 4443494)].

• Install the Enricher in a different location than the location used to prepare

emulsions containing library template and P1 Beads. Emulsification and

enrichment in different locations prevents contamination of the sample. Do not

install the Enricher and the Emulsifier in the same room. For more information,

refer to the Applied Biosystems SOLiD® EZ Bead Emulsifier Site Preparation Guide

(Part no. 4452499).

• Store unlabeled cleaning racks A and B where they can be routinely used with the

Enricher. Like the waste container, the cleaning racks are required for normal

operation of the Enricher.

14 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents

1

Prepare and install the reagents

Note: You can thaw the SOLiD EZ Bead Capture Oligo on ice as you prepare and

install reagents on the instrument (see step 6).

Note: For specific safety information on handling of butanol and butanol waste, see

“Specific alerts for chemicals” on page 84.

1. Ensure that the Enricher has been properly cleaned or primed (see Table 1).

IMPORTANT! After the Enricher has been run, always clean the instrument before

starting another enrichment run.

Table1 Clean or prime the Enricher, if necessary

2. Empty the waste bottle:

a. Remove used reagent racks A and B.

b. Empty then reinstall the waste bottle.

c. Screw on the cap with attached tubing to the waste bottle.

3. Install the Enricher Buffer Kit – Butanol:

a. Unscrew the cap with attached tubing of the previously installed bottle of

butanol.

b. Unscrew the cap of a new bottle of butanol.

c. Replace the previously installed bottle of butanol with the new bottle of

butanol in the labelled holder of the Enricher (see Figure 6 on page 18).

d. Screw the cap with the attached tubing to the new bottle of butanol so that

the cap seals the bottle.

4. Install reagent rack A on the Enricher:

a. Uncap the bottles on a rack.

b. Insert the rack on the right top railing (see Figure 5 on page 18 and Figure 6

on page 18).

c. Slide the rack until the rack is fully into the instrument and the rack stops.

5. Remove all used plastic consumables, if necessary.

6. Prepare the SOLiD® EZ Bead™ Enricher Capture Oligo:

a. Thaw the Capture Oligo on ice.

Instrument State Action

New instrument “Prime 2: Prepare the Enricher for first-

time use or after long-term storage” on

page 35.

Run just completed “Clean the Enricher” on page 27.

Preparing for long-term storage†

† Long-term storage is more than three days of disuse.

“Prime 1: Prepare the Enricher before

long-term storage” on page 32.

Instrument State Action

15

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents 1

b. Transfer 1 mL of Capture Buffer (in the SOLiD® EZ Bead™ Enricher Buffer

Kit) from bottle B1 (pink label) to the SOLiD® EZ Bead™ Enricher Capture

Oligo Tube (F lime-green tube shown in Figure 2).

Figure2 Transfer Capture Buffer to Capture Oligo Tube

c. Add 28 µL of Capture Oligo to the Capture Buffer in the Capture Oligo Tube

(F lime-green tube).

Figure3 Add Capture Oligo to Capture Buffer

d. Cap the Capture Oligo Tube and gently swirl the tube to mix the solution.

e. Install the Capture Oligo Tube into the labelled holder on the Enricher

(see Figure 5 on page 18 and Figure 6 on page 18).

f. Connect the tubing to the Luer-Lok® of the Capture Oligo Tube (F lime-green

tube).

7. Pour the Buffer Kit – CS Solution 1 Additive into bottle B4 (yellow label),

containing CS Solution 1, in the SOLiD® EZ Bead™ Enricher Buffer Kit

(see Figure 4 and Table 2).

Capture Buffer Capture Oligo Tube

Capture Oligo Capture Oligo Tube

16 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents

1

Figure4 Positions of reagent bottles on reagent racks A and B

Table2 Reagents in Reagent Racks

8. Ensure that all the bottles in Reagent Rack B are tightly capped, then mix the

reagents in the bottles by gently swirling reagent rack B for 10 seconds.

9. Install reagent rack B on the Enricher:

a. Uncap the bottles on the rack.

b. Insert the rack on the left top railing (see Figure 5 on page 18 and Figure 6 on

page 18).

c. Slide the rack until the rack is slid fully into the instrument and the rack

stops.

10. Install the tubes as listed below (see Table 3):

Rack position Reagent

A1 SOLiD® EZ Bead™ Enricher Buffer Kit – Rinse Buffer

A2 SOLiD® EZ Bead™ Enricher Buffer Kit – Hyb Buffer

A3 SOLiD® EZ Bead™ Enricher Buffer Kit – CS Solution 2

A4 SOLiD® EZ Bead™ Enricher Buffer Kit – CS Solution 3

B1 SOLiD® EZ Bead™ Enricher Buffer Kit – Capture Buffer

B2 SOLiD® EZ Bead™ Enricher Buffer Kit – Release Buffer

B3 —

B4 SOLiD® EZ Bead™ Enricher Buffer Kit – CS Solution 1

B5 —

B6 —

17

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents 1

Table3 Ensure that each tube and bottle listed below is firmly seated at the bottom of its

assigned location

a. Unscrew the tubing from the cleaning manifold.

b. Connect the tubing to the designated tubes and bottles (see Figure 5 on

page 18 and Figure 6 on page 18 and Table 3). Match the numbers/colors on

the tubing to the numbers/colors on the tubes and bottles.

c. Secure the connections with a slight, finger-tight, twist. Do not over-tighten

the connections.

Tubes to install

Matching numbers and

colors on the tubing and

tubes

__ SOLiD® EZ Bead™ Enricher QC Output Tube 5 Yellow

__ SOLiD® EZ Bead Output Tube 3 Red

__ SOLiD® EZ Bead™ Enricher Unenriched Bead Bottle 4 Black

__ SOLiD® EZ Bead™ Enricher Release Tube 11 Purple

__ SOLiD® EZ Bead Emulsifier Hyb Tube 10 Tan

__ Connector on Cleaning Manifold Connect to any other

connector on the Cleaning

Manifold

18 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents

1

Figure5 Layout of Enricher: actual view

Figure6 Layout of Enricher: schematic view

Reagent rack B

Reagent rack A

Waste bottle

SOLiD EZ Bead

Enricher Unenriched

Bead Bottle, 4 Black

Butanol

SOLiD EZ Bead

Enricher Bead Output

Tube, 3 Red, front

SOLiD EZ Bead

Enricher QC Output

Tube, 5 Yellow, middle

SOLiD EZ Bead

Enricher Capture Oligo

Tube, F Lime-green, back

SOLiD EZ

Bead Enricher

Input Tube,

8 Orange

SOLiD EZ Bead

Enricher Hyb Tube,

10 Tan

SOLiD EZ Bead

Enricher Column,

2 Blue-green, front

SOLiD EZ Bead

Enricher Capture

Bead Tube, 9 Pink,

middle

SOLiD EZ Bead

Enricher Release

Tube, 11 Purple,

back

SOLiD EZ Bead

Enricher Filter,

7 Blue

Cleaning line

Cleaning manifolds

Reagent rack B

Reagent rack A

Butanol

Waste container

SOLiD EZ Bead

Enricher Unenriched

Bead Bottle

4 Black

SOLiD EZ Bead

Enricher Capture Bead Tube

9 Pink (middle)

SOLiD EZ Bead

Enricher QC Output Tube

5 Yellow (middle)

SOLiD EZ Bead

Enricher Bead Output Tube

3 Red (front)

SOLiD EZ Bead

Enricher Column

2 Blue-green (front)

SOLiD EZ Bead

Enricher Filter

7 Blue

Cleaning line

SOLiD EZ Bead

Enricher Release Tube

11 Purple (back)

SOLiD EZ Bead

Enricher Capture Oligo Tube

F Lime-green (back)

SOLiD EZ Bead

Enricher Hyb Tube

10 Tan

SOLiD EZ Bead

Enricher Input Tube

8 Orange

Cleaning manifolds

19

Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents 1

11. Install the bottle of SOLiD® EZ Bead™ Enricher Capture Beads in the SOLiD® EZ

Bead™ Enricher Capture Bead Tube (9, pink) on the thermomixer:

a. Unscrew the cap of a new bottle of Capture Beads (stored at 4°C). Ensure that

you are using the correct bottle of Capture beads for E10, E20, E80, or E120

scale of enrichment.

b. Unscrew the cap that has a new SOLiD EZ Bead Sipping Assembly, as shown

in Figure 7.

Figure7 Unscrew cap with new Sipping Assembly

c. Screw the new Sipping Assembly on to the new bottle of Capture Beads.

Discard the used bottle that originally contained the new Sipping Assembly.

d. Place the new bottle of capture beads with the new Sipping Assembly in the

labelled location in the Emulsifier (see Figure 5 on page 18 and Figure 6).

e. Connect the tubing to the Sipping Assembly.

SOLiD EZ Bead

Sipping Assembly

Capture Beads

SOLiD EZ Bead

Sipping Assembly

Used bottle to be discarded

20 Applied Biosystems SOLiD® EZ Bead™ Enricher Getting Started Guide

Chapter1 Prepare and Run the SOLiD® EZ Bead™ Enricher

Prepare and install the reagents

1

12. Recover the emulsion:

a. Tare the SOLiD® EZ Bead™ Enricher Input Tube with its cap assembly.

b. Unscrew the cap from the pouch nozzle, and unscrew the cap assembly from

the Input Tube.

c. Pour the entire emulsion from the SOLiD® EZ Bead Amplifier Pouch into the

Input Tube.

d. Screw the cap onto the pouch nozzle.

e. Lay the pouch flat on a bench top. With a MicroAmp® Adhesive Film

Applicator, push the remaining emulsion toward the pouch nozzle.

f. Uncap the pouch nozzle, then pour the remaining emulsion into the Input

Tube.

g. Screw the cap assembly on the Input Tube.

h. Screw the cap onto the pouch nozzle, then dispose of the pouch into the

appropriate waste container.

13. Weigh the emulsion in the tared, SOLiD® EZ Bead™ Enricher Input Tube with its

cap assembly:

a. Record the weight of the emulsion for later use: _____________ g.

b. Follow the table below:

14. Place the Enricher Input Tube in the labelled location on the Enricher, then attach

the tubing to the Input Tube.

15. Install the Enricher Column (see Figure 5 on page 18 and Figure 6 on page 18 and

Figure 8):

a. Disconnect the cleaning line from the base of the Enricher Column, then

connect the cleaning line to any port on the Cleaning Manifold.

b. Unlock the clamp, if necessary.

c. Connect the tubing to the top of a new Enricher Column (see Figure 8).

d. Push the male fitting at the bottom of the Enricher column into bottom female

connection (Figure 8).

e. Clamp and lock the column.

If the weight of the

emulsion is within

range...

Then...

E10: 13 g to 18 g

E20: 22 g to 27 g

E80/E120: 100 g to 118 g

Proceed to step 14 to complete installation for the

enrichment run.

Outside the ranges

above

Do not proceed with the enrichment run. Contact

your Applied Biosystems Field Applications

Specialist.

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Thermo Fisher Scientific SOLiD™ EZ Bead™ Quick start guide

Thermo Fisher Scientific SOLiD™ EZ Bead™ Quick start guide

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