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Manual
FTD CC Light cycler 480 color compensation
Bulk for 2 color compensations (catalog no. FTD-CC-BULK)
Color Compensation
For Research Use Only
For use with the LightCycler®480 System I & II
FRUO
FTD-CC-BULK
Fast-track Diagnostics; 38, rue Hiehl; L-6131 Junglinster; Luxembourg
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
FTD Light Cycler® 480 color compensation
Table of contents
1. Identification of the manufacturer .............................................................. 3
2. Identification of the product ........................................................................ 3
3. Intended use ................................................................................................ 3
4. Contents....................................................................................................... 4
5. Precautions and warnings............................................................................ 5
5.1
Handling requirements ..........................................................................5
5.2
Safe waste disposal ...............................................................................5
6. Storage and stability conditions .................................................................. 5
7. Principle of the method................................................................................ 5
8. Additionally required equipment ................................................................. 6
9. Procedure: Create a new color compensation.............................................. 7
9.1
Pipetting a color compensation plate ......................................................7
9.2
Run a color compensation experiment on LC480 .....................................9
9.3
Create a color compensation file .......................................................... 21
9.4
Verification of the received color compensation file ............................... 22
10. Run an experiment on LC480 ................................................................... 25
11. Analysis of an experiment on LC480 ........................................................ 28
12. Legend of symbols ................................................................................... 33
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
1. Identification of the manufacturer
Fast-track Diagnostics Luxembourg S.à.r.l.
38, rue Hiehl
Z. I. Langwiss
L-6131 Junglinster
Tel. +352 780290 329
Fax: +352 780290 514
[email protected]
2. Identification of the product
FTD Light Cycler® 480 color compensation
Category:
Singleplex PCR for color compensation of
Light Cycler® 480-System I & II
Reference:
FTD-CC-BULK
Indication:
For Research Use Only
Test for 2 color compensations
3. Intended use
The Light Cycler® 480-System is able to simultaneously detect and analyze more
than one fluorescent signal in each reaction of a Real-time-PCR. As we perform
multiplex-assays with detection of two or more different dyes in one reaction,
overlapping of the emission spectra of the dyes can occur and cause the
phenomenon called “crosstalk”. This bleed-over fluorescence signal can result in
misinterpretation of data. To correct the crosstalk, color compensation (CC) can
be applied before data analysis. When the cc is activated, LC 480 Basic Software
algorithms use the data from the CC object to compensate the fluorescence
crosstalk. To obtain optimized conditions for your personal LC480 you have to
create your own color compensation with our provided FTD Light Cycler® 480
color compensation kit.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
4. Contents
Table 1: Contents of the FTD Light Cycler 480 color compensation kit.
Color
Yellow
Red
Mauve
Green
White
Label
YELLOW PP
BULK
RED PP
BULK
ORANGE PP
BULK
GREEN PP
BULK
BLANK PP
BULK
Brown
YELLOW PC
BULK
Brown
RED PC
BULK
Brown
ORANGE PC
BULK
Brown
GREEN PC
BULK
Brown
BLANK PC
BULK
Contents
Primer/Probe mix for
yellow detection
Primer/Probe mix for
red detection
Primer/Probe mix for
orange detection
Primer/Probe mix for
green detection
Primer mix for blank
value determination
Positive control
(plasmid) for yellow
detection
Positive control
(plasmid) for red
detection
Positive control
(plasmid) for orange
detection
Positive control
(plasmid) for green
detection
Positive control for
blank value
determination
Tubes for 2
color
compensations
Amount of reagent
1x
12.1 µl
1x
12.1 µl
1x
12.1 µl
1x
12.1 µl
1x
12.1 µl
1x
66 µl
1x
66 µl
1x
66 µl
1x
66 µl
1x
66 µl
The box itself, the cover of the box and each vial are labeled with a lot number.
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FTD Light Cycler® 480 color compensation
5. Precautions and warnings
5.1 Handling requirements
Use of this product should be limited to personnel trained in the techniques of
PCR. This product should be used in accordance with Good Laboratory Practice.
Take the normal precautions required for handling all laboratory reagents.
Do not mix reagents from different lots.
Do not use the product after its expiration date (shelf life 1 year).
5.2
Safe waste disposal
Dispose of unused reagents and waste in accordance with country, state or local
regulations.
6. Storage and stability conditions
The components of the FTD product should be stored in the original packaging at
–20°C and are stable until the expiration date stated on the label. The product is
shipped on deep frozen refrigerated packages which should ensure a transport
temperature under +10°C (robustness measurement, according to stability
studies). Freeze the product immediately after one column of reagents has been
taken out. Each column of reagents within a kit is suitable for two runs. More
than 10x thawing and freezing (≤10x, according to stability studies) of the
reagents per tube should be avoided, as this may reduce assay sensitivity.
For stability performance data, please refer to www.fast-trackdiagnostics.com.
7. Principle of the method
Viral nucleic acid is amplified in a polymerase chain reaction by specific primers.
Detection of product takes place via a dual-labeled molecular probes for each
virus of the multiplex PCR. The presence of specific viral sequences in the
reaction is detected by an increase in the fluorescence observed from the
relevant dual-labeled probe, and is reported as a cycle threshold value (Ct) by
the Real-time thermocycler.
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FTD Light Cycler® 480 color compensation
8. Additionally required equipment
The FTD Light Cycler 480 color compensation kit is suited for use with the
Roche-Light Cycler® 480-System I & II (Roche). The assay has been fully
validated with FTD® mastermix and AgPath-ID™ One-Step RT-PCR Kit (life
technologies). If you want to use different enzymes please refer to FTD.








Real-time PCR buffer and enzyme. We recommend FTD® mastermix or
AgPath-ID™ One-Step RT-PCR Kit (life technologies
Disposable powder-free gloves
Pipettes (adjustable)
Sterile pipette tips with filters
Vortex mixer
Desktop centrifuge
LightCycler®480 96 well PCR plates and plate sealers are recommended.
Sample rack
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FTD Light Cycler® 480 color compensation
9. Procedure: Create a new color compensation
9.1
Pipetting a color compensation plate
If you want to use different enzymes to FTD® mastermix or AgPath-ID™ OneStep RT-PCR Kit please firstly check at www.fast-trackdiagnostics.com!
Preparation of PCR with FTD® mastermix:
For pipetting of a color compensation plate, prepare a minimum of 3 (3.5)
replicate color compensation reactions for each fluorescence dye (yellow, red,
orange, green), including a blank. Each reaction (except the blank) contains one
of the detected probes.
1. Thaw reagents for the reaction:
(Reagents in each column are sufficient for 2 color compensations.)
The reagents are yellow-, red-, green-, orange- and blank-PP, the
positive controls and FTD® buffer (FTD® mastermix).
Make sure to keep the enzyme of FTD® enzyme of FTD® mastermix
cool (freezer or cooling block) at any time!
Before use, the reagents have to be thawed completely, mixed (by
pipetting or short vortexing and spinning down briefly!
2. For 1 color compensation pipette 5.25 µl of each PPmix in separate
tubes and add 43.75 µl of FTD® buffer (FTD® mastermix) to each of
them.
Take care to change the tips after each pipetting step.
3. Pipette 3.5 µl of FTD® enzyme (FTD® mastermix) to each of yellow-,
red-, green-, orange- and blank-PP.
Take care to change the tips after each pipetting step.
4. Vortex the complete master mixes briefly and spin them down in a
centrifuge.
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FTD Light Cycler® 480 color compensation
Preparation of PCR with AgPath-ID™ One-Step RT-PCR Kit:
For pipetting of a color compensation plate, prepare a minimum of 3 (3.5) replicate color compensation reactions for each
fluorescence dye (yellow, red, orange, green), including a blank. Each reaction (except the blank) contains one of the
detected probes.
1.
Thaw reagents for the reaction:
(Reagents in each column are sufficient for 2 color compensations.)
The reagents are yellow-, red-, green-, orange- and blank-PP, the positive controls and the 2xRT PCR
buffer of AgPath-ID™ One-Step RT-PCR Kit.
Make sure to keep the enzyme of the AgPath-ID™ One-Step RT-PCR Kit cool (freezer or cooling block) at
any time!
Before use, the reagents have to be thawed completely, mixed (by pipetting or short vortexing and
spinning down briefly!
2.
For 1 color compensation pipette 5.25 µl of each PPmix in separate tubes and add 43.75 µl “2xRT PCR
buffer” (AgPath-ID™ One-Step RT-PCR Kit) to each of them.
Take care to change the tips after each pipetting step.
3.
Pipette 3.5 µl of “25x RT-PCR enzyme mix” (AgPath-ID™ One-Step RT-PCR Kit) to each of yellow-, red-,
green-, orange- and blank-PP.
Take care to change the tips after each pipetting step.
4.
Vortex the complete master mixes briefly and spin them down in a centrifuge.
Table 2: Amount of reagents needed for 3.5 wells (1 color compensation). The PP in one
tube is sufficient for two color compensations plus 10% pipetting inaccuracy. The
PPmixes contain all primers and one probe. The blank reaction PP has no probe inside.
Number of reactions
Preparation
of the PCR assay
1x
3.5x
PPmix
1.5µl
5.25µl
Buffer
12.5µl
43.75µl
Enzyme
1µl
3.5µl
Total
15µl
52.5µl
Aliquot 3 times 15µl of each master mix in 3 wells, add 10 µl of the appropriate
sample in each of the wells. (please refer to the scheme below)
Blank reaction
Red reaction (Cy5)
Yellow reaction (Vic)
Green reaction (Fam)
Orange reaction (Rox)
A
B
C
D
E
F
G
H
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1 2 3 4 5 6 7 8 91 1 1
0 1 2
FTD Light Cycler® 480 color compensation
9.2
Run a color compensation experiment on LC480
Open the Light Cycler 480 Basic Software and go in “Overview” and use the
same programme settings you will use for your multicolor experiment and apply
it as a template. You will need an additional temperature gradient, refer to the
recommended programme below. You can also create a new experiment.
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FTD Light Cycler® 480 color compensation
Detection Format
Before starting the color compensation run, you have to choose the right
detection format under “Tools” with the filter combinations given below. Click on
“Detection format”.
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FTD Light Cycler® 480 color compensation
Click on “New” under “Detection Formats” and choose a name for your new
detection format. Tick the required wavelength in the “Filter Combination
Selection”. In the “Selected Filter Combination List” you can change the name of
the ticked combination after your work habits (Fam, Green or 465-510,…).
Furthermore make sure to set “Melt Factor”, “Quant Factor” and “Max
Integration Time” on 1.
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FTD Light Cycler® 480 color compensation
Be aware that the two different Light Cycler Systems I and II have different filter
combinations for the according wavelength to tick (Table 3).
Table 3: Overview of different detection formats on LC480 system I & II
Light Cycler System I
Emission Filters
500
533
Excitation
Filters
450
483
523
558
615
568
610
640
670
GREEN
YELLOW
ORANGE
RED
Light Cycler System II
Emission Filters
488
510
Excitation
Filters
440
465
498
533
618
580
610
YELLOW
ORANGE
640
660
GREEN
RED
Safe your new detection format. For each new run you are now able to choose
the new detection format under “Setup”.
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FTD Light Cycler® 480 color compensation
Instrument Setting
Please note that you have to choose the correct plate (white or clear). Make sure
to use always the same plate type for your color compensation experiment and
for your following experiments.
Programme Setting
By pressing “+” you are able to add further steps of your PCR protocol. Change
the “Progam Name”, “Cycles”, “Analysis Mode”, “Target”, “Acquisition Mode”,
“Hold”, “Ramp Rate” as provided in Table 4.
Table 4: Programme of the color compensation run.
Programme name
Cycles
Analysis mode
Retro-transcription
1
None
Pre-incubation
1
None
Amplification
40
Quantification
Temperature gradient
1
Color compensation
Cooling
1
None
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FTD Light Cycler® 480 color compensation
FTD® mastermix
Programme
name
Target
(°C)
Acquisition
mode
Hold
(hh:mm:ss)
Ramp Rate
(°C/s)
Acquisitions
(per °C)
Retro-transcription
42
None
00:15:00
4.4
-
Pre-incubation
94
None
00:03:00
4.4
-
Target
(°C)
Acquisition
mode
Hold
(hh:mm:ss)
Ramp Rate
(°C/s)
Acquisitions
(per °C)
94
None
00:00:08
4.4
-
60
Single
00:00:34
2.2
-
72
None
00:00:01
4.4
-
95
None
00:00:10
4.4
-
50
None
00:00:30
2.2
-
70
Continuous
-
0.03
5
40
None
00:00:10
2.0
-
Program name
Amplification
Temperature
gradient
Cooling
AgPath-ID™ One-Step RT-PCR Kit
Programme
name
Target
(°C)
Acquisition
mode
Hold
(hh:mm:ss)
Ramp Rate
(°C/s)
Acquisitions
(per °C)
Retro-transcription
50
None
00:15:00
4.4
-
Pre-incubation
95
None
00:10:00
4.4
-
Target
(°C)
Acquisition
mode
Hold
(hh:mm:ss)
Ramp Rate
(°C/s)
Acquisitions
(per °C)
95
None
00:00:08
4.4
-
60
Single
00:00:34
2.2
-
72
None
00:00:01
4.4
-
95
None
00:00:10
4.4
-
50
None
00:00:30
2.2
-
70
Continuous
-
0.03
5
40
None
00:00:10
2.0
-
Program name
Amplification
Temperature
gradient
Cooling
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FTD Light Cycler® 480 color compensation
FTD® mastermix
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
If you want you can start the run immediately before setting up the samples on
the “start run” button.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Sample Setting
Subset Editor:
To receive an optimal measurement and calibration of the LC480 it is necessary
to create a subset for all the well`s which are included in the color compensation
experiment. Choose the “Subset Editor” click on “+” and add the name of the
new subset. Select the correct well`s and click “Apply”.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Sample Editor:
Choose the “Sample Editor” and pick the created subset. Select “Configure
Properties”. Tick “Replicate of”, “Color Compensation” and “Dominant Channel”.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Insert “Repl of”, “Sample Name” and “Dominat Channel”.
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FTD Light Cycler® 480 color compensation
9.3
Create a color compensation file
After the run has finished, you have to click on “Analysis” on the Module bar.
From the “Create new analysis” list below, select color compensation. Choose the
created cc subset. Click OK (√). Click on “Calculate” to perform the color
compensation analysis. Confirm the analysis with “Save CC object”. By default,
the CCC folder in your “special data” folder is selected as location. Click OK (√).
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FTD Light Cycler® 480 color compensation
9.4
Verification of the received color compensation file
Click on “Analysis” and choose “Abs Quant/Fit Points” under “Create New
Analysis”. Select the Correct cc subset click OK (√).
Select “In Database” and choose the created cc. Compare all curves of different
channels with and without color compensation (Figure 1).
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FTD Light Cycler® 480 color compensation
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Without Color compensation
With Color compensation
GREEN
Yellow
Orange
red
Figure 1: Overview of verification of color compensation.
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FTD Light Cycler® 480 color compensation
10. Run an experiment on LC480
Programme and Detection Format setting
Open the Light Cycler® 480 Basic Software and create a “New Experiment” using
the recommended programme below. For further experiments you can save
these programme settings and use them later as a template. For the detailed
programme please refer to Table 5. Choose the detection format as mentioned
under 9.2 Detection format.
Table 5: LC480 FTD programme setup
PCR programme:
FTD® mastermix
AgPath-ID™ One-Step RT-PCR Kit
42°C for 15 minutes hold
94°C for 3 minutes hold
50°C for 15 minutes hold
95°C for 10 minutes hold
40 cycles of: 94°C for 8 seconds
60°C for 34 seconds
40 cycles of: 95°C for 8 seconds
60°C for 34 seconds
The PCR programme might differ if another enzyme than the recommended one
is used.
If the programme and detection format setup is finished and the plate is
inserted, the run can be started with the “start run” button. The sample setup
can take place any time after starting the run. You just have to define your
samples according to the FTD kit manual instructions.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Sample setting
Choose the “Subset Editor” click on “+” and add the name of the new subset.
Select the correct well`s and click “Apply”. Add certain subset`s according to the
amount of FTD kit`s or PPmixes you want to execute depending on your working
habits.
Choose the “Sample Editor” and pick the created subset. Select “Configure
Properties”. Tick “Quantification Analysis” if you used a quantitative test. Tick OK
(). Insert “Sample name”, “Quantification Sample Type” and for quantitative
assays the “Concentration” for quantification standards.
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FTD Light Cycler® 480 color compensation
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
11. Analysis of an experiment on LC480
Click on “Analysis” and choose “Abs Quant/Fit Points” under “Create New
Analysis”. Select the subset of interest and click OK (√). Make sure to choose the
color compensation.
We recommend to use the “Abs Quant/Fit Points” method for analysis to avoid to
miss low positive samples. In case you use the “Abs Quant/2 nd Derivative Max”
method make sure to verify uncertain samples with the “Abs Quant/Fit Points”
method.
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FTD Light Cycler® 480 color compensation
Choose the correct dye format and start to analyze. Check “Cycle Range”, “Noise
Band” and “Analysis” for baseline and threshold settings.
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FTD Light Cycler® 480 color compensation
After setting the threshold click “Calculate”. For Quantitative assays you need to
measure your standard curve by pressing “Std Curve (In run)”. The table
overview gives you Ct values and concentrations of your samples, QS1-QS3 and
NC.
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
Analyze the channel of the internal control and check the “Cycle Range”, “Noise
Band” and “Analysis” for baseline and threshold settings. Click “Calculate” and
Check the results of Ct value for your internal control.
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FTD Light Cycler® 480 color compensation
FTD cc Light Cycler 480 – MANUAL – v6 – 2014_02 EN
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FTD Light Cycler® 480 color compensation
12. Legend of symbols
Symbol
Significance
Manufacturer
Date Of Manufacture
Catalogue Number
Batch Code
Use By
Suffices For
FRUO
For Research Use Only
Temperature Limits
Caution
Consult Instructions For Use
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