1
HRM v3.0
HRM
HRM
MeltDoctor™ HRM Reagents StepOne™ Real-Time PCR
StepOnePlus™ Real-Time PCR 7500 Fast Real-Time PCR v2.0
DNA HRM Calibration File Melt
Curve
1,
2.
3.
MeltDoctor™ HRM Calibration Plate DNA
4. MeltDoctor™ HRM Calibration Plate
5. MeltDoctor™ HRM Calibration Plate
6.
MeltDoctor™ HRM
7. MeltDoctor™ HRM
8.
HRM Calibration File Melt Curve
9. HRM Calibraion File MeltDoctor™ HRM Calibration Plate
10. Melt Curve
2
HRM
96
StepOne™ Real-Time PCR
- MicroAmp® Fast Optical 48-Well Reaction Plate
- MictoAmp® Optical 48-Well Adhesive Film
StepOnePlus™ Real-Time PCR 7500Fast Real-Time PCR
- MicroAmp® Fast Optical 96-Well Reaction Plate
- MictoAmp® Optical Adhesive Film
20μL
1. 20μL
2. Optical Adhesive Film
1. Instrument Instrument Maintenance Manager
Navigation Background
2. Start Calibration Setup
3. Run START RUN
Analysis
1. Analysis
3
HRM DNA
StepOne™ Real-Time PCR
-48 HRM
>- MeltDoctor™ HRM Master Mix
- Melt Doctor™ HRM Calibration Standard (20x)
-
- MicroAmp® Fast Optical 48-Well Reaction Plate
- MictoAmp® Optical 48-Well Adhesive Film
-1000μL100μL20μL
-2mL
-
StepOnePlus™ Real-Time PCR 7500Fast Real-Time PCR
-MeltDoctor™ HRM Calibration Plate, Fast 96-Well
HRM
StepOne™ Real-Time PCR
1.
HRM
2. Instrument Maintenance Manager
3.
HRM
100μL
20X MeltDoctor™ HRM Calibration Standard
2000μL
Total volume
1000μL
2X MeltDoctor™ HRM Master Mix
900μL
4
2. MicroAmp® 48-Well Reaction Plate 20μL1.
3. Optical Adhesive Film
StepOnePlus™ Real-Time PCR 7500Fast Real-Time PCR
1. Fast 96-Well MeltDoctor™ HRM Calibration Plate
2.
1. Advanced Setup
Experiment Properties
- Experiment Name : Amplification_< >
- Experiment type : Quantitation – Standard Curve
- Reagents – SYBR® Green Reagents
-Include Melt Curve
- Ramp speed : Standard (~2 hours to complete a run)
2. Setup Plate Setup Define Targets and Samples Target 1
reporter SYBR
HRM
3. Assign Targets and Samples
a.
b. Target 1 Assign
c. Passive Reference None
c. Sample 1 Assign
5
4. Setup Run Method
-: 20μL
- :
Anneal / ExtendDenatureEnzyme ActivationStep
0:15
95
40 Cycles
6095
1:0010:00
Hold
5. HRM START RUN
6.
-HRMCalibrationFiles
-: Amplification_< >
(Experiment Name
7. Analysis
8.
1. Amplification Plot
-8 35 Threshold line
-
-C
T0.25 View Well Table
HRM
Amplification Plot
2.
6
MeltDoctor™ HRM
1. Instrument Instrument Maintenance
Manager Navigation Dye
2. Dye Custom Dye Calibration
3. Start Dye Calibration
4. Dye Library HRM
a. New Dye
b. Name MeltDoctor
c. Type Reporter
d. OK
HRM
HRM
HRM MeltDoctor™ HRM
5. Step 2 Dye Name MeltDoctor
6. Step 3 Temperature 60
7. HRM The custom
dye plate is loaded into the instrument
Next
8. Run START RUN
9. HRM Next
Analysis
1. Analysis
: HRM
2. Instrument Maintenance Manager
7
HRM Calibration File Melt Curve
1. Advanced Setup
Experiment Properties
- Experiment Name :
HRMCalibration_MeltDoctoreDye_< >
- Experiment type : Melt Curve
- Reagents – Other
- Ramp speed : Fast
2. Setup Plate Setup Define Targets and Samples Target 1
reporter MeltDoctor
HRM
Calibration File
HRM
HRM 3Melt
Curve HRM HRM Calibration File
3. Assign Targets and Samples
a.
b. Target 1 Assign
c. Passive Reference None
4. Setup Run Method
-: 20μL
- :
0:10
95
Denature
Melt Curve
AnnealHRMAnnealStep
0:15
95 6060
0:151:00
- StepOne™ / StepOnePlus™
> ramp mode Continuous ramp rate 0.3%
- 7500 Fast
> Expert Mode
> Select/View Filters Filter-1
8
5. HRM START RUN
6.
-HRMCalibrationFiles
-: HRMCalibration_MeltDoctoreDye_< >
(Experiment Name
7. Analysis
8.
1. Melt Curve Tm
Melt Curve
Melt Curve Tm PCR
2. HRM
HRM
HRM Calibration File
HRMCalibration_MeltDoctoreDye_< >
2011/05 A
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
NOTICE TO PURCHASER: PLEASE REFER TO THE HRM EXPERIMENTS USING MELTDOCTOR™ HRM REAGENTS AND HIGH RESOLUTION MELT
SOFTWARE v3.0 USER GUIDE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.
c 2010 Life Technologies. All rights reserved. The trademarks mentioned herein are the property of Life Technologies or their respective owners.
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関連論文
-
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Thermo Fisher Scientific Applied Biosystems StepOne™ StepOnePlus™ Real-time PCR Systems 取扱説明書
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-
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