Thermo Fisher Scientific SOLiD™ EZ Bead™ Quick start guide

Type
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.
© 2011 Life Technologies Corporation. All rights reserved.
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
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.
Table1 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
Table2 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
Table3 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

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Quick start guide

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