Roche LightCycler 480 Instrument Reference guide

Roche
Brand
Roche
Model
LightCycler 480 Instrument
Type
Reference guide

Roche LightCycler 480 Instrument

Below you will find brief product information for Roche LightCycler 480 Instrument:

  • Accurate and reliable - The LightCycler 480 Instrument is a real-time PCR instrument that provides accurate and reliable results. It uses a unique closed-tube system to prevent contamination and ensure that your results are accurate.
  • Fast and efficient - The LightCycler 480 Instrument is fast and efficient, with a run time of just 30 minutes. This makes it ideal for high-throughput applications.
  • Versatile - The LightCycler 480 Instrument can be used for a variety of applications, including gene expression analysis, genotyping, and pathogen detection. It is also compatible with a wide range of reagents and consumables.

Roche LightCycler 480 Instrument

Below you will find brief product information for Roche LightCycler 480 Instrument:

  • Accurate and reliable - The LightCycler 480 Instrument is a real-time PCR instrument that provides accurate and reliable results. It uses a unique closed-tube system to prevent contamination and ensure that your results are accurate.
  • Fast and efficient - The LightCycler 480 Instrument is fast and efficient, with a run time of just 30 minutes. This makes it ideal for high-throughput applications.
  • Versatile - The LightCycler 480 Instrument can be used for a variety of applications, including gene expression analysis, genotyping, and pathogen detection. It is also compatible with a wide range of reagents and consumables.
LightCycler
®
480 Instrument
Quick Guide
For life science research use only.
Not for use in diagnostic procedures.
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LightCycler® 480 Quick Guide Page i
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General Information
The contents of this document, including all graphics and photographs, are the property of Roche Diagnostics.
No part may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any
purpose, without the express written permission of Roche Diagnostics.
Roche Diagnostics has made every reasonable effort to ensure that all the information contained in this manual
is correct at the time of printing. However, Roche Diagnostics reserves the right to make any changes necessary
without notice as part of ongoing product development.
This LightCyler® 480 Instrument Quick Guide was created by the Roche Diagnostics Corporation Commercial
Education department. Direct questions or concerns regarding the contents of this document to:
Roche Diagnostics Corporation
Commercial Education Department
9115 Hague Road
P.O. Box 50457
Indianapolis, IN 46250-0457
USA
5125-00-1113
LIGHTCYCLER is a trademark of Roche. The technology for LightCycler® is licensed from Idaho Technologies,
Inc.
All other trademarks are the property of their respective owners.
©2013, Roche Diagnostics.
Distribution in USA by: Roche Diagnostics, Indianapolis, IN
This LightCycler® 480 Instrument Quick Guide is printed in the US.
Additional copies of this document can be obtained by sending a message to:
[email protected]
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Revision History
Revisions to this document are provided by Roche Diagnostics when necessary. No part of this document may be
reproduced in any form or by any means without prior written consent.
Publication Reference Number Date Revision Purpose
5125-00-1113 November 2013 New Document Creation
LightCycler® 480 Quick Guide Page 1
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LightCycler
®
480 Instrument Basics
General Required Materials
Standard Laboratory Equipment
Product Name
Microwell Plate Centrifuge
Nuclease-free, aerosol-resistant pipette tips
Microcentrifuge and 1.5 mL microcentrifuge tubes
Micropipettes (P10 or P20, P200, P1000 or equivalent)
Disposables
Product Name Catalog Number Description
LightCycler® 480 Multi-well Plate 96-well - White 04 729 692 001 50 plates and sealing foils
LightCycler® 480 Multi-well Plate 384-well - White 04 729 749 001 50 plates and sealing foils
LightCycler® 480 Multi-well Plate 96-well - Clear 05 102 413 001 50 plates and sealing foils
LightCycler® 480 Multi-well Plate 384-well - Clear 05 102 430 001 50 plates and sealing foils
LightCycler® 480 Sealing Foil 04 729 757 001 50 sealing foils
Reagents
Reaction Type Product Name Catalog Number Description
Reverse Transcription
with RNAse H activity
Transcriptor 1st Strand
cDNA Synthesis Kit
04 379 012 001 For 50 (20 µL) reactions
SYBR Green Master LightCycler® 480 SYBR
Green I Master
04 707 516 001 5 mL (5 x 20 µL)
2x concentrated Master Mix for
250 (20 µL) reactions
High Resolution
Melting
LightCycler® 480 High
Resolution Melting Master
04 909 631 001 5 mL (5 x 1 mL)
2x concentrated 25 mL MgCl2
stock solution for 500 (20 µL)
reactions
Hybridization Probes LightCycler® 480
Genotyping Master
04 707 524 001 1.5 mL
5x concentrated Master Mix for
250 (20 µL) reactions
Hydrolysis Probes
TaqMan® Probes or
Universal Probe Library
(UPL)
LightCycler® 480 Probes
Master
04 707 494 001 5 mL (5 x 1 mL)
2x concentrated Master Mix for
250 (20 µL) reactions
LightCycler® 480 Control Kit 04 710 924 001 Kit for quantitative Real-Time
PCR and genotyping reactions
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LightCycler
®
480 Instrument Basics
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LightCycler
®
480 Instrument Basics
Getting Started
1. Log on to the LightCycler® computer.
Username: Operator
Password: LC480
2. Start the LightCycler® 480 software by double-clicking the <LightCycler480> icon.
3. Enter the username and assigned password to log in to the LightCycler® 480 software.
First Time Login (new database)
Username: admin
Password: LightCycler480
Normal Login
Username: admin
Password: Roche480
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LightCycler
®
480 Instrument Basics
4. The overview screen displays. This screen allows entry of a new experiment with or without use of a template
for the conditions, or run a previously programed macro.
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LightCycler
®
480 Instrument Basics
Software Navigation Overview
Exit the soware
Logout of the current database
Switch to Overview menu
Display Navigator window
Save the current experiment
Export the current experiment to an “.ixo” le
Close the current experiment
Print the current screen
Display Tools window
Navigator window: displays the objects stored in the database as folders and files, allowing the user to work with
objects in the database as well as import and export
Exporting as .ixo: creates a copy which can be imported to another computer or database
Tools window: allows user to change passwords, edit system settings, view database status and Error Log,
define filter combination settings
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LightCycler
®
480 Instrument Basics
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LightCycler® 480 Quick Guide Page 7
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New Experiment from Template
Create a New Experiment from Template
Templates provide a convenient way to speed up the process of creating an experiment. You can use Roche general
templates for the different types of Real-time PCR reactions, as well as create your own templates based on the
specific conditions of your experiments.
1. Click New Experiment from Template in the Overview window.
2. The New Experiment from Template window displays the templates that match the connected instrument.
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New Experiment from Template
3. Select a run template.
a. Dual Color Hydrolysis Probes/UPL Probe
• duplex reactions using Hydrolysis Probes (UPL/TaqMan) labeled with FAM and VIC, HEX or
Yellow 555
b. End-point Genotyping (PCR read)
• genotyping with FAM and VIC or HEX-labeled Hydrolysis or TaqMan probes, PCR and data
collection (Allelic discrimination)
c. End-point Genotyping (Pre-Post read)
• genotyping with FAM and VIC or HEX-labeled probes, data collection only, PCR performed
in another instrument (Allelic Discrimination)
d. Gene Scanning
• basic protocol for High Resolution Melting experiments, including touch-down protocol
e. HybProbe
• reactions using Hybridization Probes, labeled with Fluorescein and LightCycler Red 640
f. Mono Color Hydrolysis Probe/UPL
• reactions with Hydrolysis Probes (UPL/TaqMan) labeled with FAM
g. Simple Probes
• melting curve genotyping reactions using a single Fluorescein-labeled probe (Simple Probe)
h. SYBR Green
• reactions using SYBR Green and simliar double-strand-intercalating dyes, includes melting
curve
4. Click . The selected run-template is applied.
5. Modify run conditions (reaction volume, number of cycles, target temperature, hold, ramp rate, etc.) as
needed.
LightCycler® 480 Quick Guide Page 9
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New Experiment from Template
6. The “Start Run” icon becomes active once the multi-well plate is loaded in the instrument.
7. Click Start Run.
8. Enter the experiment name in the dialog box.
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New Experiment from Template
Save Run Conditions as a Template
If you modify the run conditions, you can save it as a new template for future use.
1. Open or create the run to be used as a template.
2. Click the drop-down next to “Apply Template” and select Save as Template.
3. Name the run template and select a location to save the template.
Note: The default storage location for templates is the user’s Template folder.
4. Click . The template is saved and will appear in the New Experiment from Template window in the future.
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Basic Real-Time Chemistry Protocols
Typical SYBR Green Reaction Protocol
Reaction Setup
Reagent Final Concentration Volume (µL) / one reaction
Water 1.0
Primer - Forward (5 µM Stock) 0.5 µM 2.0
Primer - Reverse (5 µM Stock) 0.5 µM 2.0
LightCycler® 480
SYBR Green I Master
1x 10.0
Total 15.0
Add 5 µL template DNA for a total volume of 20 µL
Run Program
Program Name Cycles Analysis Mode
Pre-incubation 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:05:00
Program Name Cycles Analysis Mode
Amplication 45 Quantication
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:00:10
60 None
00:00:10
72 Single
00:00:10
Program Name Cycles Analysis Mode
Melting Curve 1 Melting Curves
Target (°C) Acquisition Mode Hold (hh:mm:ss) Acquisitions (per °C)
95 None
00:00:05
65 None
00:01:00
97 Continuous
5
Program Name Cycles Analysis Mode
Cooling 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
40 None
00:00:30
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Basic Real-Time Chemistry Protocols
Typical Mono Color Hydrolysis Probe (TaqMan
®
/UPL) Reaction Protocol
Reaction Setup
Reagent Final Concentration Volume (µL) / one reaction
Water 0.6
Primer - Forward (5 µM Stock) 0.5 µM 2.0
Primer - Reverse (5 µM Stock) 0.5 µM 2.0
Probe (10 µM Stock) 0.2 µM 0.4
LightCycler® 480 Probe Master 1x 10.0
Total 15.0
Add 5 µL template DNA for a total volume of 20 µL
Run Program
Program Name Cycles Analysis Mode
Pre-incubation 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:10:00
Program Name Cycles Analysis Mode
Amplication 45 Quantication
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:00:10
60 Single
00:00:30
72 None
00:00:01
Program Name Cycles Analysis Mode
Cooling 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
40 None
00:00:30
LightCycler® 480 Quick Guide Page 13
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Basic Real-Time Chemistry Protocols
Typical Hybridization Probe (HybProbe) Reaction Protocol
Reaction Setup
Reagent Final Concentration Volume (µL) / one reaction
Water 6.2
Primer - Forward (5 µM Stock) 0.5 µM 2.0
Primer - Reverse (5 µM Stock) 0.5 µM 2.0
Fluorescein Probe (10 µM Stock) 0.2 µM 0.4
Red Fluor Probe (10 µM Stock) 0.2 µM 0.4
LightCycler® 480
Genotyping Master
1x 4.0
Total 15.0
Add 5 µL template DNA for a total volume of 20 µL
Run Program
Program Name Cycles Analysis Mode
Pre-incubation 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:10:00
Program Name Cycles Analysis Mode
Amplication 45 Quantication
Target (°C) Acquisition Mode Hold (hh:mm:ss)
95 None
00:00:10
60 Single
00:00:10
72 None
00:00:10
Program Name Cycles Analysis Mode
Melting Curve 1 Melting Curves
Target (°C) Acquisition Mode Hold (hh:mm:ss) Acquisitions (per °C)
95 None
00:01:00
40 None
00:02:00
95 Continuous
5
Program Name Cycles Analysis Mode
Cooling 1 None
Target (°C) Acquisition Mode Hold (hh:mm:ss)
40 None
00:00:30
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Basic Real-Time Chemistry Protocols
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LightCycler® 480 Quick Guide Page 1
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Absolute Quantification
Absolute Quantification Analysis
Sample concentrations are calculated based on a standard curve created with standard samples along a
concentration range with specific assigned values. The standard curve can be included within the run (internal
standard curve) or generated in a previous run (external standard curve).
Important: To use an external standard curve imported from a previous run, the software needs a
reference point included in the current run. One concentration standard must be on every run
to align the standard curve into the new run. The software shifts the imported standard curve
based on how the reference standard compares to the previously run concentration.
For more information, please consult Chapter D, Section 4.2.3 (pages 164-166) of the
LightCycler® 480 Operator’s Manual.
Crossing point (Cp) values (Cp is equivalent to Ct) are used to determine unknown sample concentrations from a
standard curve.
Performing an Absolute Quantification Analysis
1. Open the experiment you want to analyze in the main window.
Note: Once the experimental run has completed, it remains open and ready to analyze. Previous
experiments are located in the Experiments folder of the Navigator.
2. If sample information was entered during run setup, proceed to step 5. Otherwise, click Sample Editor on the
left side of the active display.
3. In Sample Editor > Step 1: Select Workflow, select AbsQuant workflow.
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Absolute Quantification
4. Enter Sample information
a. In Sample Editor > Step 2: Select Samples, select wells.
b. In Sample Editor > Step 3: Edit Abs Quant Properties, enter the appropriate sample information for
the selected wells. Identify samples as “Unknown” or “Standard” samples, entering the appropriate
concentration for each standard.
c. For reactions in replicate, select all the wells and click “Make Replicates.”
5. Click Analysis.
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Roche LightCycler 480 Instrument Reference guide

Roche
Brand
Roche
Model
LightCycler 480 Instrument
Type
Reference guide

Roche LightCycler 480 Instrument

Below you will find brief product information for Roche LightCycler 480 Instrument:

  • Accurate and reliable - The LightCycler 480 Instrument is a real-time PCR instrument that provides accurate and reliable results. It uses a unique closed-tube system to prevent contamination and ensure that your results are accurate.
  • Fast and efficient - The LightCycler 480 Instrument is fast and efficient, with a run time of just 30 minutes. This makes it ideal for high-throughput applications.
  • Versatile - The LightCycler 480 Instrument can be used for a variety of applications, including gene expression analysis, genotyping, and pathogen detection. It is also compatible with a wide range of reagents and consumables.
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