Mx3000P & Mx3005P Real-Time QPCR System MxPro Software version 4.10 Revision# 090530
Mx3000P & Mx3005P Real-Time QPCR System 1
PCR Stratagene SYBR Green I PCR Brilliant II FAST SYBR QPCR Master Mix Cat# 600843 400 Brilliant II SYBR Green QPCR Master Mix Cat# 600828 400 Brilliant II SYBR Green QPCR Low ROX Master Mix Cat# 600830 400 Brilliant II SYBR Green QRT-PCR Master Mix, 1-step Cat# 600825 400 Brilliant II SYBR Green QRT-PCR Low ROX Master Mix, 1-step Cat# 600835 400 Brilliant II SYBR Green QRT-PCR AffinityScript Master Mix, 2-step Cat# 600834 400 Brilliant SYBR Green Core Reagent Kit Cat# 600546 400 PCR Brilliant II FAST QPCR Master Mix Cat# 600845 400 Brilliant II QPCR Master Mix Cat# 600804 400 Brilliant II QPCR low ROX Master Mix Cat# 600806 400 Brilliant II QRT-PCR Master Mix, 1-step Cat# 600809 400 Brilliant II QRT-PCR low ROX Master Mix, 1-step Cat# 600837 400 Brilliant II QRT-PCR AffinityScript Master Mix, 2-step Cat# 600827 400 Brilliant II QRT-PCR Core Reagent Kit, 1-step Cat# 600810 400 Brilliant Multiplex QPCR Master Mix Cat# 600553 200 SideStep SideStep SYBR Green QPCR Master Mix Cat# 400904 400 SideStep II SYBR Green QRT-PCR Master Mix, 2 Step Cat# 400909 400 SideStep II QRT-PCR Master Mix, 1-step Cat# 400917 400 SideStep II QRT-PCR Master Mix, 2-step Cat# 400918 400 SideStep Lysis and Stabilization Buffer Cat# 400900 10 ml SideStep Cell Lysate Analysis Kit Cat# 400916 100 SideStep II QPCR cdna Synthesis Kit Cat# 400908 50 cdna & AffinityScript QPCR cdna Synthesis Kit Cat# 600559 50 AffinityScript Multiple Temperature Reverse Transcriptase Cat# 600107 50 Mx3000P & Mx3005P Real-Time QPCR System 2
RNA Stratagene QPCR Reference Total RNA, Human Cat# 750500 25µg Stratagene QPCR Reference Total RNA, Mouse Cat# 750600 25µg Universal Human mirna Reference RNA Cat# 750700 10µg mirna High-Specificity mirna QRT-PCR Detection Kit Cat# 600580 200 mirna QPCR Master Mix Cat# 600583 200 mirna QRT-PCR First strand cdna Synthesis Kit Cat# 600036 50 High-Specificity mirna QPCR Core Kit Cat# 600545 200 QPCR Alien QRT-PCR Inhibitor Alert Cat# 300600 400 Alien Reference RNA QRT-PCR Detection Kit (FAM-detection) Cat# 300602 100 Alien Reference RNA QRT-PCR Detection Kit (VIC-detection) Cat# 300604 100 RNA Absolutely RNA Miniprep Kit Cat# 400800 50 Absolutely RNA Microprep Kit Cat# 400805 50 Absolutely RNA Nanoprep Kit Cat# 400753 50 Absolutely RNA 96 Microprep Kit Cat# 400793 2 plates Absolutely mrna Purification Kit Cat# 400806 10 Absolutely RNA FFPE Kit Cat# 400809 50 Absolutely RNA mirna Kit Cat# 400814 50 Mx3000P Mx3005P Mx3000P / Mx3005P Replacement Bulb Assembly Cat# 401411 1 bulb 96-Well QPCR Plate, Semi-Skirted Cat# 401334 25 plates 96-Well QPCR Plate, Non-Skirted Cat# 401333 25 plates Mx3000P / Mx3005P Strip tubes Cat# 401428 120 strips Mx3000P / Mx3005P Optical Strip Caps Cat# 401425 120 strips Single PCR Reaction Tubes (Without Caps) Cat# 410023 1 bag MxP Perfect Frame Cat# 401421 1 frame Mx3000P & Mx3005P Real-Time QPCR System 3
Brilliant master mix Brilliant 2.5 ml Reference dye (ROX 1mM) 0.5µl ROX Reference dye DW 200 ROX 20µl 0.3µl ROX 20µl SYBR Green 2x Master Mix 10µL 10µL Primer xµl xµl Probe none xµl Template xµl xµl DW Up to 20µL Up to 20µL Mx3000P/Mx3005P PCR 10µl 60µl standard curve QPCR Mx3000P/Mx3005P Mx3000P & Mx3005P Real-Time QPCR System 4
Mx3000P/Mx3005P / Mx3000P/Mx3005P ON Mx3000P/Mx3005P PC ON Mx3000P/Mx3005P 96 well 8 Optical cap Mx3000P & Mx3005P Real-Time QPCR System 5
8 96 well / Mx3000P/Mx3005P Mx3000P / Mx3000P & Mx3005P Real-Time QPCR System 6
MxPro Ver.4.10 MxPro Mx Pro Multiple Experiment Analysis Mx3000P/ Mx3005P PC PC Quantitative PCR (Multiple Standards) TaqMan probe Molecular Beacon Standard curve Mx3000P Mx3005P QPCR Dissociation curve Comparative Quantification (Calibrator) Ct Standard TaqMan probe SYBR Green Dissociation curve Standard curve QPCR SYBR Green (with Dissociation Curve) SYBR Green Dissociation curve Mx3000P & Mx3005P Real-Time QPCR System 7
EvaGreen (with Dissociation Curve) SYBR Green EvaGreen Stratagene High Specificity mirna Detection kit Allele Discrimination / SNP s Real-Time Molecular Beacon amplification plot SNP Molecular Beacon Melting Curve Molecular Beacon Quantitative Plate Read Standard curve SYBR Green PicoGreen RiboGreen EvaGreen Plate Read / Allele Discrimination TaqMan probe SNP Multiple Experiment Analysis Standard curve Mx3000P & Mx3005P Real-Time QPCR System 8
Mx Pro Setup Run Analysis Multiple Experiment Analysis Project Setup Setup Plate Setup Thermal Profile Thermal Profile Run Run Thermal Profile Status Raw data plots Mx3000P/Mx3005P Analysis Analysis Analysis Selection / Setup Results Project Project Project Setup Project Results Mx3000P & Mx3005P Real-Time QPCR System 9
Plate Setup Experimental type Quantitative PCR (Multiple Standards) SYBR Green (with Dissociation curve) Import mxp Defaults Quick Setup Mx3000P & Mx3005P Real-Time QPCR System 10
Well type Well type Show Well Names Well Information Well Name Collect fluorescence data Mx3000P/Mx3005P MxPro Mx3000P/Mx3005P Reference dye Reference dye Assign Assay Names Well Information Well name Comments Assign Standard Quantity Standard template Auto Increment Standard Units Standard template Identify replicates Replicate symbol replicate well Threshold line Minimum Ct spread algorithm replicate symbol Clear Selected Wells Mx3000P & Mx3005P Real-Time QPCR System 11
Comparative quantification (Calibrator) Well type Comparative quantification Calibrator Normalizing assay Ct Association symbol SYBR Green Normalizer Unknown Normalizer Association symbol Mx3000P & Mx3005P Real-Time QPCR System 12
Undo Redo On / Off MxPro ver.4.1 ON 20 PC MxPro Replicate Wells treatment setting Analysis Term Setting Threshold line Baseline Amplification plot Chart Mx3005P Mx3000P Mx3000P & Mx3005P Real-Time QPCR System 13
Plate Setup Quick Protocol Mx3000P & Mx3005P Real-Time QPCR System 14
Plate Setup Quick Protocol (SYBR Green ) DNA SYBR Green I PCR SYBR Green PCR PCR standard template 10 6 10 10 3 NTC Sample # Gene Well type Filter Conc. Well 1 A unknown SYBR A1-A3 2 A unknown SYBR B1-B3 3 A unknown SYBR C1-C3 4 B unknown SYBR A4-A6 5 B unknown SYBR B4-B6 6 B unknown SYBR C4-C6 7 A standard SYBR 10 6 A7-A9 8 A standard SYBR 10 5 B7-B9 9 A standard SYBR 10 4 C7-C9 10 A standard SYBR 10 3 D7-D9 11 A NTC SYBR E7-E9 12 B standard SYBR 10 6 A10-A12 13 B standard SYBR 10 5 B10-B12 14 B standard SYBR 10 4 C10-C12 15 B standard SYBR 10 3 D10-D12 16 B NTC SYBR E10-E12 Mx3000P & Mx3005P Real-Time QPCR System 15
MxPro SYBR Green SYBR Green (with Dissociation Curve a) # Well type unknown Mx3000P & Mx3005P Real-Time QPCR System 16
b) unknown SYBR Collect fluorescence data SYBR FAM ROX ROX Reference dye ROX Filter SYBR Collect fluorescence data FAM SYBR FAM Mx3000P/Mx3005P PC PC Mx3000P/Mx3005P PC Mx3000P & Mx3005P Real-Time QPCR System 17
SYBR Green FAM SYBR SYBR SYBR Green FAM FAM SYBR Green SYBR ROX REF c) Well A4 C6 d) #7 #11 standard NTC Mx3000P & Mx3005P Real-Time QPCR System 18
standard #7 #10 well A7 D9 e) Well type Standard Standard f) SYBR ROX ROX Reference dye Mx3000P & Mx3005P Real-Time QPCR System 19
g) #11 NTC well E7 E9 Well type NTC SYBR ROX ROX Reference dye h) 12 16 (e) (g) Mx3000P & Mx3005P Real-Time QPCR System 20
i) Replicate symbol well A1 A3 well B1 B3 Replicate symbol Replicate #1 well A1 A3 Replicate Symbol Auto-increment Replicate symbol Mx3000P & Mx3005P Real-Time QPCR System 21
j) copies Standard Units Copies #7 #12 10 6 copies Mx3000P & Mx3005P Real-Time QPCR System 22
A7 A12 Auto-Increment -10x Auto-Increment Standard quantity SYBR SYBR all Auto-Increment Auto- Increment 10 6 copies 1/10-10 #8 #13 10 5 copies Auto-Increment Auto-Increment Mx3000P & Mx3005P Real-Time QPCR System 23
k) triplicate standard Replicate #1 #3 #7 #11 A Well Information SYBR Assay Assay Color Assay A Assay Color Mx3000P & Mx3005P Real-Time QPCR System 24
#4 #6 #12 #16 B Mx3000P & Mx3005P Real-Time QPCR System 25
Plate Setup Quick Protocol (Probe ) TaqMan probe Molecular Beacon PCR MxPro Quantitative PCR (Multiplex Standards) TaqMan probe Molecular beacon Simple assay TaqMan probe PCR FAM TaqMan probe A VIC TaqMan probe B standard template 10 6 10 10 3 NTC Mx3000P & Mx3005P Real-Time QPCR System 26
Sample # Gene Well type Fileter Conc. Well 1 A unknown FAM A1-A3 2 A unknown FAM B1-B3 3 A unknown FAM C1-C3 4 B unknown HEX A4-A6 5 B unknown HEX B4-B6 6 B unknown HEX C4-C6 7 A standard FAM 10 6 A7-A9 8 A standard FAM 10 5 B7-B9 9 A standard FAM 10 4 C7-C9 10 A standard FAM 10 3 D7-D9 11 A NTC FAM E7-E9 12 B standard HEX 10 6 A10-A12 13 B standard HEX 10 5 B10-B12 14 B standard HEX 10 4 C10-C12 15 B standard HEX 10 3 D10-D12 16 B NTC HEX E10-E12 a) # A unknown triplicate Well type unknown Mx3000P & Mx3005P Real-Time QPCR System 27
b) unknown FAM TaqMan probe Collect fluorescence data FAM ROX ROX Reference dye ROX FAM ROX REF Mx3000P & Mx3005P Real-Time QPCR System 28
c) FAM TaqMan probe VIC TaqMan probe FAM HEX VIC HEX Well A4 C6 d) #7 #11 standard NTC standard #7 #10 well A7 D9 Mx3000P & Mx3005P Real-Time QPCR System 29
e) Well type Standard Standard f) FAM ROX ROX Reference dye g) #11 NTC well E7 E9 Well type NTC FAM ROX ROX Reference dye Mx3000P & Mx3005P Real-Time QPCR System 30
h) 12 16 (e) (g) FAM HEX i) Replicate symbol well A1 A3 well B1 B3 Replicate symbol Mx3000P & Mx3005P Real-Time QPCR System 31
Replicate #1 well A1 A3 Replicate Symbol Auto-increment Replicate symbol j) FAM HEX 12 [all] 12 Mx3000P & Mx3005P Real-Time QPCR System 32
Standard quantity Standard units Standard units copies relative milimole Standard quantity FAM HEX Standard quantity all Replicate #7(well A7 A9) Replicate #13 (well A10 A12) standard all Auto-Increment -10x Auto-Increment Standard Mx3000P & Mx3005P Real-Time QPCR System 33
FAM A HEX (VIC) B SYBR Green Well information FAM FAM standard standard curve FAM HEX (VIC) Well information FAM HEX (VIC) Well information Well information FAM SYBR Green Eva Green FAM SYBR Green unknown standard Well information Well information SYBR Green Mx3000P & Mx3005P Real-Time QPCR System 34
Multiplex assay FAM HEX VIC Multiplex Sample # Gene Well type Filter Conc. Well 1 A/B unknown FAM & HEX A1-A3 2 A/B unknown FMA & HEX B1-B3 3 A/B unknown FAM & HEX C1-C3 4 A/B standard FAM & HEX 10 6 A4-A6 5 A/B standard FAM & HEX 10 5 B4-B6 6 A/B standard FAM & HEX 10 4 C4-C6 7 A/B standard FAM & HEX 10 3 D4-D6 8 A/B NTC FAM & HEX E4-E6 Multiplex a) unknown A1 C3 Well type unknown FAM HEX ROX Reference dye ROX Mx3000P & Mx3005P Real-Time QPCR System 35
b) Standard well A4 - D6 Well A4 D6 Well type standard FAM HEX ROX Reference Dye ROX c) NTC Well E4 E6 Well E4 E6 Well type NTC FAM HEX ROX Reference Dye ROX d) Replicate symbol Mx3000P & Mx3005P Real-Time QPCR System 36
e) Standard Standard Units Standard Quantities Auto-Increment Simple Multiplex assay Well information Well information Multiplex assay Mx3000P & Mx3005P Real-Time QPCR System 37
Plate Setup Quick Protocol ( ) SYBR Green SYBR Green A B (b-actin) A B RSq NTC 20µl 3 Sample # Gene Well type Filter Description Well #1 A calibrator SYBR A1-A3 #2 A unknown SYBR B1-B3 #3 A unknown SYBR C1-C3 #4 B calibrator SYBR A4-A6 #5 B unknown SYBR B4-B6 #6 B unknown SYBR C4-C6 #7 b-act calibrator SYBR A7-A9 #8 b-act unknown SYBR B7-B9 #9 b-act unknown SYBR C7-C9 Mx3000P & Mx3005P Real-Time QPCR System 38
Sample # Gene Well type Filter Conc. Well #10 A standard SYBR 1 x D1-D3 #11 A standard SYBR 1/10 x E1-E3 #12 A standard SYBR 1/100 x F1-F3 #13 A standard SYBR 1/1000 x G1-G3 #14 A NTC SYBR H1-H3 #15 B standard SYBR 1 x D4-D6 #16 B standard SYBR 1/10 x E4-E6 #17 B standard SYBR 1/100 x F4-F6 #18 B standard SYBR 1/1000 x G4-G6 #19 B NTC SYBR H4-H6 #20 b-act standard SYBR 1 x D7-D9 #21 b-act standard SYBR 1/10 x E7-E9 #22 b-act standard SYBR 1/100 x F7-F9 #23 b-act standard SYBR 1/1000 x G7-G9 #24 b-act NTC SYBR H7-H9 MxPro Comparative Quantification (Calibrator) Quantitative PCR SYBR Green Comparative Quantification Mx3000P & Mx3005P Real-Time QPCR System 39
a) #1 #1 well A1 A3 A Calibrator Well A1 A3 well type Calibrator Comparative quantification Multiplex 50 SYBR ROX Reference dye ROX Well A1 A3 SYBR Green p17 p18 Mx3000P & Mx3005P Real-Time QPCR System 40
b) #2 #3 #2 #3 well B1 C3 A Unknown a Well B1 C3 Well type Unknown SYBR ROX Reference dye ROX c) #4 B Calibrator #5 #6 B Unknown Reference dye A d) normalizer #7 #9 a c #7 b-actin Calibrator #8 #9 b-actin Unknown Mx3000P & Mx3005P Real-Time QPCR System 41
e) #10 #13 Standard #14 NTC #10 #13 Well D1 G3 Well type Standard SYBR ROX Reference dye ROX Well H1 H3 #14 NTC Well H1 H3 Well type NTC (No Template Control) SYBR ROX ROX Reference dye Mx3000P & Mx3005P Real-Time QPCR System 42
f) #15 #19 B Standard NTC #20 #24 b-actin Standard NTC e #15 #19 Well D4 H6 #20 #24 Well D7 H9 g) Replicate symbol Association symbol Mx3000P & Mx3005P Real-Time QPCR System 43
h) #1 #3 #10 #14 A Well information A #1 #3 #10 #14 Well A1 Well H3 Well information SYBR SYBR Assay A Assay Color Mx3000P & Mx3005P Real-Time QPCR System 44
A #4 #6 #15 #19 B #7 #9 #20 #24 b-actin Well information Mx3000P & Mx3005P Real-Time QPCR System 45
i) Standard #10 #15 #20 Well D1 Well D9 Standard quantity 1 #11 #15 #21 1/10 #12 #17 #22 1/100 #13 #18 #23 1/1000 Auto Increment Standard Mx3000P & Mx3005P Real-Time QPCR System 46
j) b-actin Normalizer Normalizer Well type b-actin #7 #9 Calibrator #7 #7 Well A7 A9 Normalizing assay b-act Well A7 A9 b-act NORM Unknown #8 #9 Normalizer Normalizer Well type Calibrator Unknown Mx3000P & Mx3005P Real-Time QPCR System 47
normalizer MxPro version 4.10 Normalizer BRCA Replicate #1 4 GAPDH Replicate #5 8 18S Replicate #9 12 GUS Replicate #13 16 Normalizer Normalizing assay GAPDH 18S GUS Normalizer Ct Ct Normalizer Mx3000P & Mx3005P Real-Time QPCR System 48
k) Association #1 A #4 B #7 b-act Well A1 A9 Well A1 A9 Assoc. Symbol A A Well B1 B9 Well C1 C9 B C Mx3000P & Mx3005P Real-Time QPCR System 49
Standard Association symbol Standard Standard curve Relative quantity to Standard Multiplex Cy5 b-actin FAM A VIC B normalizer b-actin association A FAM B VIC Multiplex b-actin SYBR Green Association Mx3000P & Mx3005P Real-Time QPCR System 50
normalizer Multiplex normalizer LIMS MxPro version 4.10 LIMS (Laboratory Information Management System) LIMS LIMS ASCII Mx3000P & Mx3005P Real-Time QPCR System 51
Thermal Profile Setup Quick Protocol Quantitative PCR (Multiple Standards) Comparative Quantification (Calibrator) Thermal Profile SYBR Green Eva Green Dissociation curve SYBR Green Comparative quantification Dissociation curve Dissociation curve profile Quantitative PCR Comparative Quantification Experimental type Thermal Profile Thermal Profile Thermal profile Thermal Profile Design Standard Pre-Melt / RT Segment(s) Amplification Segment Dissociation / Melt Segment Mx3000P & Mx3005P Real-Time QPCR System 52
Thermal Profile Design Custom Add Segment Plateau with Ramp Data collection marker by dragging All point End point Thermal Profile Plateau Ramp Ramp Ramp 0.2 scan Ramp Dissociation curve Plateau Ramp Plateau Selection Plateau Ramp Segment Property Import Thermal Profile Import Custom Defaults Thermal Profile Mx3000P & Mx3005P Real-Time QPCR System 53
Thermal Profile Plateau Plateau Ramp Segment Segment Plateau Thermal Profile Data collection marker Data collection marker Thermal Profile Data collection marker Plateau Ramp Ramp All point Data collection marker Ramp 0.2 scan Mx3000P & Mx3005P Real-Time QPCR System 54
Running the Experiment Quick Protocol PCR Lamp Ready Lamp Start-up Run RUN Run Status Stopped Mx3000P/Mx3005P Turn lamp off at end of run Start Run Run Status Stopped Mx3000P/Mx3005P Warming Up 20 Run After Warm-Up 20 Run Now 20 Thermal Profile Mx3000P & Mx3005P Real-Time QPCR System 55
Raw Data Plots Raw Data Plots Raw Data Plots Show Plots well Analysis X Y Raw Data Plots Mx3000P & Mx3005P Real-Time QPCR System 56
Mx3000P/Mx3005P Run Status Running Add Cycle Thermal Profile PCR Run Status Add cycle Pause Run Pause Thermal profile Run Run Continue Continue E-mail experiment at end of run Run Mx3000P/ Mx3005P PC Mx3000P & Mx3005P Real-Time QPCR System 57
Data Analysis Quick Protocol Analysis / Selection Setup Quantitative PCR (Multiple standard) Analysis Analysis Selection / Setup Select data Collection ramp / plateau Exclude Assays Mx3000P & Mx3005P Real-Time QPCR System 58
Replicates Treat individually Treat collectively Algorithm enhancements Amplification-based threshold Adaptive baseline Moving average Analysis Term Setting Analysis Term Setting Adv. Algorithm Settings Data Collection Marker Average SYBR Green Select data collection ramp/plateau: Dissociation curve Dissociation Data Dissociation curve Mx3000P & Mx3005P Real-Time QPCR System 59
Results Results Analysis / Selection Setup Customized Tool bar Mx3000P & Mx3005P Real-Time QPCR System 60
Amplification plots Area to analyze Show Standard Curve Amplification plot Standard curve Last cycle Amplification plot endpoint Fluorescence R (Multicomponet view) dr Rn drn Threshold fluorescence Threshold line Threshold line Threshold line Analysis / Selection Setup Threshold line Select amplification plots to display Mx3000P & Mx3005P Real-Time QPCR System 61
Fluorescence R (Multicomponent) dr Rn drn Show Standard Curve Amplification plot Standard curve Standard curve RSq Eff amplification plot threshold line RSq Eff Mx3000P & Mx3005P Real-Time QPCR System 62
Plate sample values Ct Standard Curve Show Amplification Plots Standard curve Amplification Plots Show confidence interval: confidence interval Threshold fluorescence Standard curve threshold line Initial template quantity calculator Ct Amplification efficiency calculator Slope Eff Mx3000P & Mx3005P Real-Time QPCR System 63
Initial template quantity standard curve Ct Text Report File Power Point Mx3000P & Mx3005P Real-Time QPCR System 64
amplification plot amplification plot Options Preferences Preferences Text Report tooltips Mx3000P & Mx3005P Real-Time QPCR System 65
amplification plot Ctrl Shift Show Amplification Plots Mx3000P & Mx3005P Real-Time QPCR System 66
Consolidated Report Consolidated report Dissociation Curve (for SYBR or Eva Green Assays) Dissociation curve SYBR Green Eva Green Experimental type Results Quantitative PCR Experimental type Thermal profile Dissociation curve Results Dissociation curve Experimental type SYBR Green Experimental type Dissociation curve Dissociation curve Experimental Type SYBR Green Eva Green Results Dissociation curve For ramp dissociation use cycle: Cycle increment Dissociation curve Ramp All point Fluorescence Mx3000P & Mx3005P Real-Time QPCR System 67
R (Multicomponent view) Rn -R (T) -Rn (T) Product melting temperature (Tm) Tm Min peak height Dissociation curve Tm Experimental type Experimental type File Convert Experimental Type Mx3000P & Mx3005P Real-Time QPCR System 68
Relative quantity (Comparative quantification ) Comparative quantification Experimental type Relative quantity_chart Relative quantity_plate Relative quantity chart Calibrator Ct Show fold change log2 Fold change Show error bar Graph Properties X Y p74 Mx3000P & Mx3005P Real-Time QPCR System 69
Relative quantity plate Calibrator Text report Calibrator Standard Rel. Quant. to Std) Normalizer (house keeping gene ) Rel. Quant. to Norm. Mx3000P & Mx3005P Real-Time QPCR System 70
Ct MxPro Analysis Term Setting Efficiency Setting 100% Normalizer Target standard curve Standard curve Efficiency Setting Eff Eff = Efficiency Mx3000P & Mx3005P Real-Time QPCR System 71
Threshold line Baseline Moving average Analysis Term Setting Threshold line Threshold Fluorescence Baseline Baseline Correction Baseline Moving average Smoothing Moving average Mx3000P & Mx3005P Real-Time QPCR System 72
Graph Properties Amplification plot Dissociation curve Standard curve Dual color plot Relative quantity chart Graph Properties X axis Y axis Ledgend On/Off Bkgd colors Style X-Axis Label Plate Setup Well ID Mx3000P & Mx3005P Real-Time QPCR System 73
Multiple experiments analysis MxPro Ver.4.10 Mx3000P Mx3005P Thermal profile Exp.1.mxp unknown Standard curve Exp.2.mxp unknown Exp.1.mxp Standard curve Exp.2.mxp Exp.1.mxp Exp.2.mxp amplification plot Ct threshold line Standard curve Mx3000P & Mx3005P Real-Time QPCR System 74
MxPro New Options Multiple Experiment Analysis Multiple Experiment Analysis Mx3000P/Mx3005P PC Standalone Multiple Experiment Analisys Add Experiments Finish Remove Experiments Multiple experiment analysis threshold line MxPro ver.4.10 Multiple experiment analysis threshold line Mx3000P & Mx3005P Real-Time QPCR System 75
threshold line Method 1) Calculate thresholds for individual experiments method threshold line new project Background-Based Threshold Algorithm Amplification-Based Threshold Algorithm Backgroung-Based Threshold Algofithm threhshold line method Data view type By Experiment threhshold value threhshold line By Assay Consolidated Data view type threshold line threshold line Background based Analysis Term Setting Threshold Fluorescence Background-based threshold Cycle range Background-based threshold Mx3000P & Mx3005P Real-Time QPCR System 76
Backgroung-based threshold threshold line amplification plot ROX threshold line Setup amplification plot Threshold fluorescence threshold line Lock threshold line threshold line standard curve amplification plot ROX standard IRC (Inter-run control) Method 3 threshold line Method 2) Use common threshold method threhshold line MxPro Project assay threshold line threshold line Ct Data view type By Experiment, By Assay, Consolidated threshold line Mx3000P & Mx3005P Real-Time QPCR System 77
Method 3) Use interrun calibrators Interrun calibrator (IRC) Run Ct threshold line Multiple experiment analysis Hellemans et.al., Genome Biology, 2007; 8(2): R19 IRC Well Id IRC Select IRC Wells IRC Multi Experiment Analysis (1) Well name (2) Assay name Well information Well name Assay name Mx3000P & Mx3005P Real-Time QPCR System 78
Multi Experiment Analysis Project Project Setup Setup Run Analysis Areas per page Project Results Project Results Area to analyze Data view taype By Experiment By Assay Consolidated Mx3000P & Mx3005P Real-Time QPCR System 79
Amplification plot amplification plot Fluorescence drn dr Setup Plate sample value Ct Mx3000P & Mx3005P Real-Time QPCR System 80
Standard curve Consolidated Amplification plot Ct Standard curve Consolidated By Experiment By Assay Initial template quantity Standard curve Text report Columun Mx3000P & Mx3005P Real-Time QPCR System 81
MxPro Ver.4.10 Normalizer Multiple Experiment Analysis Normalizer 3 samples Multiple experiment analysis threshold line Comparative quantification Multiple experiment analysis threshold line p77 Mx3000P & Mx3005P Real-Time QPCR System 82
house keeping gene A B C Inter-run control (Standard) Normalizer Mx3000P & Mx3005P Real-Time QPCR System 83
A B C Normalizer Comparative quantification Experiment type Convert A B C Normalizer Well information Well Name Well name Mx3000P & Mx3005P Real-Time QPCR System 84
Replicate #1 Sample 01 Name Sample 01 A B C Name Sample 01 Assay GAPDH sample 02 sample 06 Standard Show Well Names Well Name Mx3000P & Mx3005P Real-Time QPCR System 85
GAPDH Normalizer Calibrator Replicate #1 sample 01 Calibrator Association symbol Sample Normalizer Name Mx3000P & Mx3005P Real-Time QPCR System 86
Show Well Names Name Assign Assay Name Well information Name Assay Name Association symbol Multi Experiment Analysis Association symbol Invalid Multi Experiment Analysis Multi Experiment Analysis Relative quantity chart Standard NTC Project Standard curve Mx3000P & Mx3005P Real-Time QPCR System 87
standard curve normalizer Multiple experiment analysis normalizer Threshold line threshold line Project Cross experiment threshold calculation Use interrun calibrators Interrun Calibrators Mx3000P & Mx3005P Real-Time QPCR System 88
Project Project Results Project Amplification plot By assay By experiment Consolidated Assay per page Mx3000P & Mx3005P Real-Time QPCR System 89
Standard curve Relative quantity chart Mx3000P & Mx3005P Real-Time QPCR System 90
Multiplate Comparative assay Mx3000P & Mx3005P Real-Time QPCR System 91
Quantitative Plate Read Analysis Quick Protocol Set Read properties SNP Thermal cycling Set Read Properties Instrument data Plate Read Start Options Pre-Read Post-Read Pre-Read Post-Read Pre Post Run Status - Stopped Mx3000P & Mx3005P Real-Time QPCR System 92
Plate Setup PCR Well type Unknown Standard NTC Collect fluorescence data Reference dye ROX Reference dye Assign Assay Name Well information Standard quantity Standard Replicate symbol Replicate Import Quick Setup Mx3000P & Mx3005P Real-Time QPCR System 93
Run Plate Setup Read properties 25 99 RUN Start Run Pre-Read Post-Read Pre-Read Post-Read Mx3000P & Mx3005P Real-Time QPCR System 94
Raw data plot Raw data plot Raw data Raw data Show Plots Mx3000P & Mx3005P Real-Time QPCR System 95
Analysis Selection/Setup 1 Shift Ctrl 2 individually collectively Adv.Algorithm Settings Analysis Results Analysis Selection/Setup Plate sample values Standard curve Initial template quantity Dual color scatter plot Final call results Text report Consolidated reports Plate sample values well Ct Standard curve Standard well Ct Unknown well Mx3000P & Mx3005P Real-Time QPCR System 96
Dual color scatter plot well Ct Final call results well (+) (-) Final call Ct Ct (+) Text report Column Mx3000P & Mx3005P Real-Time QPCR System 97
Mx3000P/Mx3005P Allele discrimination/snp s Mx3000P/ Mx3005P QPCR TaqMan probe Allele discrimination / SNP s MxPro Real time PCR Advanced End point Allele discrimination/snp New Options Allele Discrimination / SNP s Real-Time Plate Read / Allele Discrimination Allele Discrimination / SNP s Real- Time TaqMan probe SNP/Mutation A/G SNP/Mutation SNP/Mutation TaqMan probe A T TaqMan probe FAM G C TaqMan probe VIC DNA PCR SNP/Mutation SNP/Mutation TaqMan probe A-homozygote FAM G-homozygote VIC heterozygote FAM VIC Dual color plot Negative control (NTC) NTC Mx3000P & Mx3005P Real-Time QPCR System 98
Allele Discrimination/SNP s Real-Time unknown: triplicate FAM VIC TaqMan probe SNP MxPro SNP/Mutation NTC (No template control) NTC FAM probe VIC probe SNP NTC FAM VIC Mx3000P/Mx3005P VIC HEX HEX VIC 20µl 3 Sample # Well type Filter Description Well #1 VIC positive FAM/VIC VIC positive cont. A1-C1 #2 FAM positive FAM/VIC FAM positive cont D1-F1 #3 unknown FAM/VIC Patient #1 A2-C2 #4 unknown FAM/VIC Patient #2 D2-F2 #5 unknown FAM/VIC Patient #3 A3-C3 #6 unknown FAM/VIC Patient #4 D3-F3 #7 unknown FAM/VIC Patient #5 A4-C4 #8 unknown FAM/VIC Patient #6 D4-F4 #9 NTC FAM/VIC No template A5-C5 SNP PCR 96 well plate / tubes Mx3000P / Mx3005P MxPro Mx3000P & Mx3005P Real-Time QPCR System 99
Allele Discrimination / SNP s Real-Time OK VIC positive (Allele 1) FAM positive (Allele 2) Unknown NTC Plate setup a) VIC HEX HEX VIC Optics Configration Mx3000P & Mx3005P Real-Time QPCR System 100
HEX-JOE Filter HEX VIC HEX VIC b) Well A1 C1 VIC positive c) Well A1 B1 C1 Well type VIC positive d) VIC FAM ROX ROX Reference dye Mx3000P & Mx3005P Real-Time QPCR System 101
e) D1 F1 FAM positive Well A2 F4 unknown Well A5 C5 NTC f) Replicate symbol Replicate Symbol Auto-increment Mx3000P & Mx3005P Real-Time QPCR System 102
g) Thermal profile Thermal profile Tm Thermal profile PCR h) Real-Time PCR Run Run PCR p55 Mx3000P & Mx3005P Real-Time QPCR System 103
Plate Read / Allele Discrimination SNP PCR 96 well plate / tubes Mx3000P / Mx3005P MxPro Plate Read / Allele Discrimination OK VIC positive (Allele 1) FAM positive (Allele 2) Unknown NTC Plate setup Plate Setup Allele Discrimination / SNP s Real-Time Plate Read Amplification plot End point Mx3000P/Mx3005P PCR Mx3000P/Mx3005P Thermal cycler Mx3000P & Mx3005P Real-Time QPCR System 104
Plate Read Mx3000P/Mx3005P Run Pre-Read Post-Read Mx3000P & Mx3005P Real-Time QPCR System 105
MxPro SNP MxPro Allele Discrimination /SNP s Allele Discrimination / SNP s Real-Time Plate Read / Allele Discrimination Plate Read / Allele Discrimination Allele Discrimination / SNP s Real-Time Amplification plot Area to analyze Mx3000P & Mx3005P Real-Time QPCR System 106
Allele Discrimination / SNP s Real Time Amplification plot Real-Time PCR a) Analysis Selection/Setup b) Results Amplification plot threshold line Results Amplification plot Amplification plot FAM VIC amplification plot FAM amplification plot FAM VIC homozygote FAM- Negative Threshold line Mx3000P & Mx3005P Real-Time QPCR System 107
negative VIC amplification plot FAM homozygous VIC negative negative threshold line Threshold line FAM VIC amplification plot VIC FAM c) Dual color plot Mx3000P & Mx3005P Real-Time QPCR System 108
Dual color plot amplification plot Last cycle Last cycle allele discrimination Dual color plot Amplification plot Dual color plot Amplification plot Show Dual clor plor line Last cycle Plate Read / Allele Discrimination Plate Read / Allele Discrimination experiment type SNP PCR SNP Real-time Negative threshold line End point Dual color plot Real-time PCR End point Mx3000P & Mx3005P Real-Time QPCR System 109
Dual color plot Real-Time PCR Plate Read Dual Color Scatter Plot Dual Color Scatter Plot allele Dual Color Scatter Plot FAM TET FAM TET FAM TET A allele FAM G allele TET A homozygote G homozygote heterozygote wild-type mutant-type Dual color plot plot Allele discrimination positive control NTC plot Mx3000P & Mx3005P Real-Time QPCR System 110
Plate sample value Real-Time Last cycle Dual color plot Amplification Plot Display the values for Ct Plate Read Fluorescence Rpost Rpre Dual color scapter plot Genotype Rectangle Mx3000P & Mx3005P Real-Time QPCR System 111
Allele associations Rename Alleles/Genotypes Allele Rename Alleles/Genotypes Dual color scatter plot Genotype rectangle Rectangle Reset genotype Rectangle Show all genotype Genotype Mx3000P & Mx3005P Real-Time QPCR System 112
confidence Final Call Result VIC FAM NTC positive Text Report Final Call Mx3000P & Mx3005P Real-Time QPCR System 113
Allele Discrimination / SNP s Real-Time Experiment Type Molecular Beacon probe Molecular beacon SNP allelic composition allele Molecular beacon allele molecular beacon A/G SNP A G Allele A allele molecular beacon Molecular beacon (MB) Molecular beacon + matched oligo (MBO) Molecular beacon + mismatched oligo (MBMO) Molecular beacon Molecular Beacon Melting Curve Experiment Type MBO MBMO MB MBO Molecular beacon 65 Molecular beacon MBMO Molecular beacon SNP MBO 50 Mx3000P & Mx3005P Real-Time QPCR System 114
SNP 60 MBO MBMO MBO MBMO 55 PCR threshold line Minimum Ct Spread algorithm MBMO amplification plot MBMO Ct MBO MBMO SNP allele Molecular beacon Molecular beacon FAM + FAM-MBO TET-MBO TET + FAM-MBMO TET-MBMO Mx3000P & Mx3005P Real-Time QPCR System 115
FAM TET FAM-MBMO TET-MBMO threshold line FAM TET Molecular beacon Ct FAM A-allele TET G-allele Molecular beacon heterozygote FAM+ heterozygote TET+ Heterozygote Molecular beacon unknown Threshold line Mx3000P & Mx3005P Real-Time QPCR System 116
FAM positive(homozygote) TET positive(homozygote) Heterozygote Dual Color Plot FAM-homozygote Heterozygote Negative TET-homozygote Amplification plot SNP endpoint amplification plot Molecular beacon Melting curve MxPro Mx3000P & Mx3005P Real-Time QPCR System 117
Molecular Beacon Melting Curve Analysis Quick Protocol Analysis Selection/Setup 1 Shift Ctrl 2 Select data collection ramp/plateau 3 individually collectively Adv.Algorithm Settings 4 Results 5 Area to analyze Annealing range (MBO) (MBMO) Melting curves/temperature Tm Mx3000P & Mx3005P Real-Time QPCR System 118
Beacon Designer http://www.premierbiosoft.com/molecular_beacons/index.html or http://www.premierbiosoft.com/stratagene/stratagene.html Beacon Designer Windows Mac CD Mac MacOSX safari MS IE Mx3000P & Mx3005P Real-Time QPCR System 119
Mx3000P Mx3005P 1000 2000 Cat# 401411 Mx3000P Real-Time PCR System Bulb 48,000 2008 Mx3000P / Mx3005P OFF Mx3000P / 3005P Mx3000P & Mx3005P Real-Time QPCR System 120
1) 2) 3) 4) 5) 6) 7) 8) 9) Lamp Reset and Utilities Mx3000P & Mx3005P Real-Time QPCR System 121
Dye and Quencher choice Efficiency = [10 (-1 / Slope) ]-1 MxPro PCR Mx3000P & Mx3005P Real-Time QPCR System 122
Stratagene s Mx3000P / Mx3005P Realtime QPCR system 192-8510 e-mail jtech@agilent.com Mx3000P & Mx3005P Real-Time QPCR System 123