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Summary of Contents for Fluidigm Juno
- Page 1 PN 100-7074 D1 Genotyping with Juno Getting Started Guide...
- Page 2 © 2017 Fluidigm Corporation. All rights reserved. 09/2017 For technical support visit fluidigm.com/support. North America +1 650 266 6100 | Toll-free (US/CAN): 866 358 4354 | techsupport@fluidigm.com Latin America +1 650 266 6100 | techsupportlatam@fluidigm.com Europe/Middle East/Africa/Russia +44 1223 859941 | techsupporteurope@fluidigm.com Japan +81 3 3662 2150 | techsupportjapan@fluidigm.com...
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Page 3: Table Of Contents
Contents About This Guide Obtain the Minimum Required Genomic DNA Prepare the Sample Mix Purpose Load and Run the IFC on Juno How to Use This Guide Perform Genotyping Analysis on the Samples Safety Alert Conventions Safety Alerts for Chemicals... -
Page 4: About This Guide
With high-quality samples, detecting the specific targets requires thermal cycling for preamplification and PCR for genotyping on the instrument. After genotyping is performed on the Juno system, the IFC is scanned on the ™ system or the Biomark ™... -
Page 5: How To Use This Guide
How to Use This Guide The chapters in this guide are organized according to assay type. Refer to the appropriate chapter to run the Juno 96.96 Genotyping IFC on the Juno system. For detailed instructions on instrument and software operation, refer to the Juno System User Guide (PN 100-7070). -
Page 6: Safety Data Sheets
Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals ordered from Fluidigm Corporation, either alone or as part of this system, go to fluidigm.com/sds and search for the SDS using either the product name or the part number. -
Page 7: Getting Started
Store reagents at their specified storage temperatures. (See “Required Kit Contents” on page 8.) • Vortex reagents for 20 seconds, and then centrifuge reagents for 2 seconds before use. Related Documentation Go to fluidigm.com/documents Genotyping with Juno Getting Started Guide... -
Page 8: Chapter 1: Product Information
Chapter 1: Product Information Required Kit Contents The kits include the reagents required for preparing 10 IFCs to use on the Juno system. For suggested kits, see “Suggested Kits” on page 28. IMPORTANT • Do not pipet reagents from the TaqMan and SNPType assay kits into the same IFC. -
Page 9: Snp Type Assay Kit
20 syringes/ Juno GT Flux Fluid 60X SNP Type Reagent Dilution Reagent Juno SNP Type GT Master Mix Juno GT Preamp Master Mix Figure 2. Juno™ SNP Type Genotyping Kit for 10 IFCs (PN 100-8364). Genotyping with Juno Getting Started Guide... -
Page 10: Required Reagents
SNP Type assays ASP1/ASP2 Fluidigm — (100 μM each) SNP Type assays LSP Fluidigm — (100 μM each) Suggested Reagents Product Name Company Part Number UltraPure ™ DNase/RNase-Free Distilled Thermo Fisher 10977-015 Water Scientific Genotyping with Juno Getting Started Guide... -
Page 11: Required Consumables
® PN 1402-9700) Required Equipment Product Name Company Part Number Juno system, including system software version v3.1 or Fluidigm 101-6455 later, instrument, software, MX Interface Plate, Interface Plate Loading Fixture, Cleaning Plate, and Barrier Tape Applicator and Adapter For Juno 96.96 Genotyping IFC: SX Interface Plate... -
Page 12: Required Software
Chapter 1: Product Information Required Software Required Software • Fluidigm Data Collection software v4.2 or later • Fluidigm SNP Genotyping Analysis software v4.2 or later IFC Type and Related Scripts Barcode (prefix) Scripts Description 180x Juno 96.96 Fast Preamplification and genotyping of samples by... -
Page 13: Chapter 2: Genotyping With The Juno 96.96 Genotyping Ifc Using Taqman Assays
— * See step 1. The final concentration of each primer in the preamplification reaction is 45 nM. NOTE The volume can be adjusted proportionally based on the number of samples to be amplified. Genotyping with Juno Getting Started Guide... -
Page 14: Prepare 2X Taqman Assays For Genotyping
Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays Prepare Assay and Sample Mixes Prepare 2X TaqMan Assays for Genotyping If necessary, adjust the concentration of TaqMan genotyping assays with DNase-free water to 18 μM (20X). In a new 96-well plate, dilute the 20X TaqMan genotyping assays in Dilution... -
Page 15: Prepare The Sample Mix
Prepare the Sample Mix In a DNA-free hood, in a new 1.5-mL microcentrifuge tube labeled “Sample Pre-Mix,” combine the Juno GT Preamp Master Mix and the primer pool for preamplification to prepare the sample pre-mix. (See Table 3.) Label a new 96-well plate “SAMPLE PLATE.” Pipet 2.25 μL of the sample pre-mix into each well of the plate. -
Page 16: Load And Run The Ifc On Juno
Load and Run the IFC on Juno Load and Run the IFC on Juno Review the loading map, which is affixed to the bottom of every new Juno 96.96 Genotyping IFC. The loading map is a general guide to show you how to pipet... - Page 17 Ensure that the notched corner of the IFC (“A1”) is at the top left. Load an entire syringe of Juno 96.96 GT Control Line Fluid in Acc1 and a second syringe in Acc2. (See pink squares on the pipetting map.) To ensure correct accumulator volume, only use syringes containing Juno 96.96 GT Control Line Fluid.
- Page 18 Carefully dispense control line fluid into the reservoirs. If control line fluid comes into contact with the sample inlets, use a new IFC. Pipet 15 μL of Juno GT Flux Fluid into each of the six ports. (See purple circles on the pipetting map.) Unseal the TaqMan assay plate and pipet 4.0 μL of each assay mix into an assay...
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Page 19: Perform Genotyping Analysis On The Samples
Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays Perform Genotyping Analysis on the Samples After the run is finished, tap EJECT to eject the IFC. IMPORTANT After a run, perform an end-point read of the IFC in ≤1 hour. Do not leave the IFC overnight in the instrument. -
Page 20: Chapter 3: Genotyping With The Juno
Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays Prepare Assay and Sample Mixes Prepare the 200 nM Primer Pool for Preamplification In a new 1.5-mL microcentrifuge tube, combine 2 μL of 100 μM SNP Type assays specific target amplification primers (100 μM STA) up to a total of 96 assays. -
Page 21: Prepare 2X Snp Type Assays
Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays Prepare Assay and Sample Mixes Prepare 2X SNP Type Assays Prepare 50X Primer Mix for Each Single Assay Inlet In a DNA-free hood, in a new 96-well plate, combine the following reagents for each... -
Page 22: Prepare The Assay Mix
Prepare the Assay Mix In a DNA-free hood, in a new 1.5-mL microcentrifuge tube labeled “Assay Pre-Mix,” combine the Juno SNP Type GT Master Mix and 60X SNP Type Reagent to prepare the assay pre-mix. (See Table 5.) Label a new 96-well plate “SNP TYPE ASSAY PLATE.” In a DNA-free hood, pipet 2.5 μL of the assay pre-mix into each well. -
Page 23: Prepare The Sample Mix
Prepare the Sample Mix In a DNA-free hood, in a new microcentrifuge tube labeled “Sample Pre-Mix,” combine the Juno GT Preamp Master Mix and the primer pool for preamplification to prepare the sample pre-mix. (See Table 6.) Label a new 96-well plate “SAMPLE PLATE,” and then pipet 2.25 μL of the sample pre-mix into each well of the plate. -
Page 24: Load And Run The Ifc On Juno
Load and Run the IFC on Juno Load and Run the IFC on Juno Review the loading map, which is affixed to the bottom of every new Juno 96.96 Genotyping IFC. The loading map is a general guide to show you how to pipet... - Page 25 Carefully dispense control line fluid into the reservoirs. If control line fluid comes into contact with the sample inlets, use a new IFC. Pipet 15 μL of Juno GT Flux Fluid into each of the six ports. (See the purple circles on the pipetting map.)
- Page 26 Ensure that the SX interface plate (silver label) is installed in the instrument. [See the Juno System User Guide (PN 100-7070).] Place the IFC into the Juno instrument, then start the run <60 minutes after pipetting the reagents into the IFC.
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Page 27: Perform Genotyping Analysis On The Samples
Chapter 3: Genotyping with the Juno 96.96 Genotyping IFC Using SNP Type Assays Perform Genotyping Analysis on the Samples Table 8 SNP Type genotyping Cycles Temperature Time Hot start 95 ºC 10 min 95 ºC 15 sec 64 ºC 45 sec 72 ºC... -
Page 28: Appendix A: Suggested Kits
60X SNP Type Reagent Amber 2 tubes 70 μL Reagent Kit (PN 100-8363) Juno GT Flux Fluid Purple 1 tube 1.0 mL Dilution Reagent Natural • 2 bottles • 3.7 mL • 1 tube • 1.7 mL Genotyping with Juno Getting Started Guide... -
Page 29: Suggested Reagents To Use With Taqman Assay And Snp Type Assay Kits
Juno 96.96 Genotyping IFC — 1 IFC — Room Genotyping IFC temperature (PN 100-6499) Juno 96.96 GT Juno 96.96 GT Control Line — 20 syringes — Room Control Line Fluid Fluid temperature (PN 100-8574) Genotyping with Juno Getting Started Guide... -
Page 30: Appendix B: Safety
For more information, see the BMBL guidelines at: cdc.gov/biosafety/publications/index.htm For a full list of the symbols on the instrument, refer to the Juno System User Guide (PN 100-7070). Genotyping with Juno Getting Started Guide... -
Page 31: Electrical Safety
Appendix B: Safety Electrical Safety Electrical Safety WARNING ELECTRICAL HAZARD. Electrical shock can result if the Juno instrument is operated without its protective covers. WARNING ELECTRICAL HAZARD. Plug the system into a properly grounded receptacle with adequate current capacity. Chemical Safety Read and comprehend all safety data sheets (SDSs) by chemical manufacturers before you use, store, or handle any chemicals or hazardous materials. - Page 32 Back Cover 7000 Shoreline Court, #100 South San Francisco, CA T: 650 266 6000 For technical support visit fluidigm.com/support.