Fluidigm Access Array, User Manual

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide
3
Contents
About This Guide 5
Safety Alert Conventions 5
Safety Data Sheets 6
Chapter 1: Access Array System
Overview 7
Access Array System Components 10
Access Array IFCs 11
Specifications 11
Access Array System Workflow 12
Materials 12
Required Reagents 12
Required Consumables 13
Required Equipment 14
Example Sample and Primer Layout in 96-Well
Plates
14
Before You Begin 15
Chapter 2: Designing Targeted
Sequencing Primers 16
Chapter 3: Target-Specific Primer
Validation for 4-Primer Amplicon
Tagging on the LP 48.48 IFC 17
Reference Documents 17
Materials 17
Required Reagents 17
Prepare Primer Validation Reactions 18
Set Up the PCR Reactions in a 384-Well PCR
Plate
20
Run the PCR Reactions 20
Check PCR Products on the Agilent 2100
Bioanalyzer
21
Chapter 4: Sample Quantitation and
Normalization 22
Reference Documents 22
Materials 22
Required Reagents 22
Required Equipment 22
Sample Quantitation 23
Sample Normalization 23
Chapter 5: Bidirectional Amplicon
Tagging on the LP 48.48 IFC 24
Reference Documents 25
Materials 25
Required Reagents 25
Prime the IFC 26
Prepare the 20X Primer Solutions 28
Prepare Sample Pre-Mix and Samples 29
Prepare the Sample Pre-Mix Solution 29
Prepare the Sample Mix Solutions 29
Load the IFC 30
Thermal-Cycle the IFC 31
Harvest the IFC 31
Attach Sequence Tags and Sample Barcodes 33
Prepare the Sample Pre-Mix Solution 33
Prepare a 100-Fold Dilution of the Harvested PCR
Products
33
Prepare the Sample Mix Solutions 33
Thermal-Cycle the 96-Well PCR Plate 34
Check PCR Products on the Agilent 2100
Bioanalyzer
35
Next Step 35
Chapter 6: Post-PCR Amplicon
Purification and Quantitation 36
Reference Documents 36
Materials 36
Required Equipment 36
Required Reagents 36
Quantitate PCR Products 37
Purify Harvested PCR Products 37
PCR Product Library Quantitation Procedure 38
Agilent 2100 Bioanalyzer Quantification 38
PicoGreen Fluorimetry Quantification 39
Pool the Products from Multiple Access Array
IFCs
39