LightCycler® Nano System ユーザートレーニングガイド, バージョン 1.0
Contents
1. LightCycler Nano System Roche Diagnostics GmbH I MM Roche Diagnostics GmbH Sandhofer Stra e 116 68305 Mannheim Germany AS 105 0014 2 6 1 TEL 03 5443 5287 mi BE II LIGHTCYCLER MAGNA PURE RESOLIGHT FASTSTART ProbeLibrary LNA Vedbaek Exiqon A S SYBR Molecular Probes Inc VIC2. 2 SNP 5 1 PCR Q EoNFXYO EISTa 7 EB HT WEPCRIIP REISE CIR gt DNA 20 10 gt NTC 20 2 ul ana DNA 90 ng ul 20 ng 5 ERS TUT A
3. LightCycler Nano Instrument LED Roche Diagnostics GmbH Mannheim Germany Made in U CE MARK CE MARK HOT SURFACE WEEE LightCycler Nano Instrument
4. 17 Q Samples a Run Settings data E Samples Targets Color Name Note db Color Name Reference SamplesasPlate Targets as Plate Note Sample e Samples Q RZEZI TZET Samplet 45 Samplesn gt F COS Bl CU ZJL E 4 EZ EZ L CX Fla Color Name Note lt E 2 11 16 GL DLORER Sg7p e7 Sample1 1 Samples Color Name Note qp Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 ES Sample 8 Sample 9 Sample 10 Sample 11 gt gt gt EZI A EU PCROZIP SY PLE LESIONS SEXIER
5. Samples 23 YLT OfgfE EIUS C ORE TS ARC JESU 6 Intensity RFU 5 10 15 20 25 30 35 40 45 Cycle Color by Sample v Dye SYBR Green I v Show all dyes results Results as Table Samples as Plate Cq Values as Plate Results as Table Results as Table CA BiG AR Y ZA AELN REAAPELZVaecCalli eU X3 RIO YT VOC Cq Unkn A 4 Unkn Results as Table Samples as Plate Cq Values as Plate Pos Note Excl Sample SYBR Green Type Cq Quantity Al 1 BE Target Js A2 2 StdDNA 1E1 Target1 is 10 32 091 A 10 001 A3 3 Target1 S 100 28 86 A 91 873 A4 4 T
6. http technical support roche com VII LightCycler Nano Instrument Version 1 0 AAT RICA S ASA PT Quantification Cycle C eooo i eewo o EP Endpoint Fluorescence LENNEENL T MEN C m Polymerase Chain Reaction Protection Earth qPCR PCR Relative Fluorescence Unit SD Standard Deviation Single Nucleotide Polymorphism SYBR SYBR Green wooo mam O lI Universal ProbeLibrary cm ZEBLO SBE
7. gt Targeti gt 1X10 42 2 P Std 1 gt Inout 7 gt gt Target gt Wells as Table Targets as Plate A6 B6 A7 amp B7 P Unk Color Name Note 4 Color Name Dye i Reference Targeti StdDNA 1E1 Target2 SYBR Green StdDNA 1E2 StdDNA 1E3 StdDNA 1E4 Unkn A Unkn B Set Number All Clear Std V Unk m Neg Clear Samples as Plate Targets as Plate l
8. TH TEHL JU ETE UCV e RHEL BI A EGE BEL TEZ RIKI WI BETEO S ERU IB OT HEDIS RPAIIEEYSccCC SUTS LightCycler Nano Instrument Version 1 0 LightCycler Nano Instrument D 1 LightCycler Nano Instrument LightCycler Nano Instrument LightCycler Nano Instrument LightCycler Nano Instrument e LightCycler Nano Instrument POST VAF LAOH PI Light Cycler Nano Software z l 2 LightCycler Nano Software
9. heart kidney liver heart b 2 Color Name Note dh EH kidney ped liver Om Ho e 23 FOTN OM EELY 4 c P Sample gt Wells as Table Samples as Plate A1 A2 B1 B2 C1 C2 D1 D2 gt Set Color Name Color Name Dye Reference heart kidney liver 9 Set Number All Clear 8 Std y Unk p Neg Clear Pos Note Sample Al A2 A3 A4 A5 AB AT A8 A c BEES c BONS EGGS M es O 3 58 Wells as Table Samples as Plate
10. 2 Q UZ hO F SINE S O d heL C ERRLEF KI TULEK OK hel TREES AIT JHZXIUCR I SUTLZMAESEGUX T Sample Sample30 b NTC NTC2 Samples Color Name Note Sample 1 Ta Sample 2 lv Sample 3 Sample 4 Sample 5 Samnle A PUD IEDTIVCEIW Y CED 288 VO H DIL OMEEL 4 EZ BRER gt Sample Wells as Table Samples as Plate A1 gt Se7 Name Note Color Name Reference CL Sample 2 Ez Sample 3 Ee Sample 4 Sample 5 9 Number All Clear 8 Std y Unk n Neg mms Clear Wells as Table ESF ii Samples as Plate Targets as Plate Pos Note Sample 70
11. Enapoint 207 results Thresholds Cycles Allele 1 Allele1 v Allele 2 Allele2 v Results as Table Samples as Plate Results as Plate EPF 1 Bample7 Q Ctl al Results as Table results Results as Table CH amp f amp 47 Samples as Plate Results as Plate 7L h Cf SBF 4 2 kBees d E ERS T AAE RA E nA ia ORE Results as Table Results as Table Call gt Allelel Allele 1 47e e Allele 2 Heterozygous Results as Table
12. Analysis 30 E EIE BZ Automatic Quantification method Select Analysis P Select Analysis pe gt High Resolution Melt Se ec7 m Select Analysis Type Wy Automatic Quantification Absolute and relative quantification using automatic CQ calling Fit Point Quantification Absolute and relative quantification using fit point CQ calling Tm Calling Melt temperature analysis ya Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping Spectra View Display offull spectrum optical data Analysis Raw Settingst Raw Raw Settings Samp les Normalization Settings Temp Shift Settings Difference Notes
13. LightCycler Nano Instrument gt LightCycler Nano Instrument b Complete gt Data En ava Gee Er PCROZIP SY PLE LES INCI SHEXTES EM TT i ORE 1 3 Automatic Quantification Automatic Quantification method eb Analysis UL Profile Data Analysis Select Analysis e Select Analysis Select Analysis 0e Select Analysis 4 q LightCyc
14. gt Target2 Exclude Standards Efficiency 2 Quantifiers Selected Target Target Lv Efficiency 2 Exclude Standards B Import Export gt Heference Reference2 Analysis Stages Relative Quantification results Sg77O es gt Calibrator Sg7p e2 OO Targets Analysis Results Color Name Note Calibrator kidney La liver M H20 d ACafB0ds Sy T7 7 J 2 7 y tTiehnac3Uc AACE S Hn 698 790 82171248 p DIRE C STR ze gt Bar by samples by targets gt Bar Chart Type Ratio Normalized ratio Relative Quant
15. FAM 1x10 1x10 10 3 1 1 10 4 6 LightCycler Nano Instrument 9 1 1 20 PCR 32 Q ey hy FORD REF 2 7 LIK ENTER HY PIVEPCRS vZCUBSSILEST CO ERU PCR TM dre
16. e DNA 20 ul amp LightCycler 8 Tube Strips clear 2 NTC 20 pl Strip A Strip B Strip C Strip D bi Q do BALSA To SZLEEMBEEL TEAL S EJET ORM PEP RET SU 1 96 MWP 17 zb MWP 3 000 x g 30 LightCycler Nano Instrument F Version 1 0 EROFAIAS gt PERI 5 2 Q XB EVIVZCE TOZIOR BOwucd 18 70 5X807HZ73vZCSEfT OJSEC S RES E AN 9 2 1 18 VAN Run Settings 2 c Bie x 3 Optics Settings
17. Analysis Stages Absolute Quantification Relative Quantification Settings Quantifiers Amplification Amplification Selected Target Target MM Excluded early cycles Min Relative Amp Intensity RFU Min Amp Quality 5 10 15 20 25 30 35 40 45 Cvcle Color by Sample vj Dye FAM v Show all dyes Results as Table Samples as Plate Cq Values as Plate Pos Note Excl Sample FAM Type CH Quantity Al 1 M u 32517 A 1 323478 A2 2 32 735 A 1 150 81 A3 3 32452 A 1 380 238 A4 4 32117 A 1 711 377 A5 5 33194 A 856 296 AB 6 33 396 A 752 278 AT 7 t i A8 8 25000 28 116 A 22 414 135 2 gt gt gt EZI A E 2 5 y h QMEBEY TD LER 2 BREA LF VL ERCE BMRB LORE e Quantifiers Target Quantifiers Target Target E Cq Settings ouantmers Amplification Selected Target Target z te 5 Cq 1 5 2 2 3 sm 4 4 5 Loa o q Cq 3 58 logie q 43 7 r 0 9984 Error 0 16 E 1 902 Int Exclude Standards Import Z Export
18. LightCycler Nano Instrument Microsoft Windows 7 LightCycler Nano Software e P a LightbyclertE Mana sy 1 0 gt Start 3 0 LightCycler Nano LightCycler Nano SW 1 0 P Nano exe 7 LightCycler Nano Software LightCycler Nano Instrument N Version 1 0 Light Cycler Nano Software D Mac OS X 10 6 LightCycler Nano Software ae e gt Finder Applications Nano
19. Program Temp C Ramp C s Hold s T Profile e Programs 20x Z0 Temperature oe Temperature Profile Temperature C 0 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 Time minutes seconds Name 2 Step Amplification Hold 95 C for 600s No of Cycles 45 Ej Temp CC Ramp Cis Hold s Acquire UTE TELM NAAN 2 step amplification 45 cycles of 95 C to 60 C 60 4 40 A de Add Delete Up 3 Down dPAdd Delete Au WW Down LightCycler Nano Instrument F Version 1 0 X 4 4h2ZBEIXCUZERLILADPEXZCHUTBeUZL s tSUNBAWES XERDUDHZZIDLZUCX T 3 2 3 Sg77O es
20. Sg7jp es Thresholds Cycles Endpoint Graph 3 Allele 1 Allele1 v Allele 2 Allele2 M Results as Table Pos Note Excl Sample Call EPF 1 EPF 2 1 1 Sample 1 7 96 A2 2 Sample 2 Allele 1 94 558 1 544 A3 3 La 68 49 136 535 Ad 4 La 7 881 238 431 AS 5 us Allele 1 90 893 2 16 AG 6 Sample 6 59 665 146 813 AT p 51 Sample 7 9 832 251 359 Ag 8 Li Allele 1 99 275 1 239 nz n no 4 gt gt gt Er AN LET Thresholds 27 Endpoint Graph gt Endpoint Graph TUES 6 Q LIDAR IIA 2 OMOVOE TWO fe ltAllele 7 Allele2 ONT OEL CHHANET Thresholds Endpoint Graph Cycles Min Angle 30 k T Min Radius 0 1 p NTC no amplification NA PAIS Thresholds
21. gt LightCycler Nano Software b AC LightCycler Nano Instrument gt LightCycler Nano Instrument LightCycler Nano Instru ment QQ e title bar X VUA Iq NU E LightCycler Nano Instrument N Version 1 0 1 A Absolute Quantification YD ices 31 43 65 Amplification Fit Point Fit point Quantification 50 Automatic Quantification 33 45 51 Analysis Results oo 67 Analysis high resolution melting s 77 81 Tae n RR ae NE DO DO i 93 95 30 33 48 45 48 51 1
22. LightCycler Nano Instrument LightCycler Nano Instrument I IEC AC PE 115 230 V PE
23. different plot curve Fs 4 1 PCR Q ArT eio LISTER PCR gt DNA 20 10 NTC 20 2 DNA 90 ng ul 20 ng 5 En ava Gee High Hesolution Melting e DNA 20 ult LightCycler 8 Tube Strips clear NTC20 Strip A Strip B Strip C Strip D bi Q SEICAUSN CU SZECHRBL CCEA SERNZBIU CU ANES ORN PILAE DAND EET Zeb SIL T ai 2ARUJZ EE096XM
24. FAM Hydrolysis Probes 62 gt gt Normal Q g7 Optics Settings Please select the brightest dye present in any well If you are unsure use the Intercalating Dyes option Intercalating Dyes SY BR Green i ResoLighi Hydrolysis Probes FAM WiC HEX Yellow 555 Red 610 TexasRed Cy5 e Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality m High Speed The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity Ifthe option is not selected the same settings selected above will be used throughout the run Optimize Melt Acquisitions Use Advanced Settings Display Dye Calibrations E X Tuskh2ESCTUZERLLADJXSTANAVCTR FUZL sE tSOHXMAES EXBOD HZTSIeZctEfr 3 2 2
25. A das gt uL CEL Cl SIZE ERP Bim ZOACEPBWET A A rm BEbBLUP Gaia LightCycler Nano SystemlCId RwNZ ZIC FERE CEB YTAILI DP SEN WEF LBLEYETI IAD FENN dL ELIE BORED EUVISCEECMHMBEC EA WAIL AZEBEZI N UTJEA PATON FBGENCLSRREEIML YA Otg A GER YI OBH aFICLEPIEAPEIFENES CIA BD Da OFF IEE BUCO CE CEIULEVA ID SRB DRESS Tet MELET LightCycler Nano Instrument
26. Wells as Table Samples as Plate Targets as Plate 1 2 3 4 5 6 7 8 Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray uw X 4 4hABEIXCUZERLIAPEXZCUtTBerUZL s tSUNBWES FROP OIF TERI 58 standard F negative unknown gt A1 A4 B1 B4 C1 C4 D1 D4 Samplel amp Sample2 gt A5 A6 B5 B6 C5 C6 D5 D6 5gzp e 2 4 aysys 67 fd JOE OH Unknown A7 B7 C7 D7
27. 3 3 1 4 PCR Q LYRE YT OWL KIEF TIKES PCR PCR 4 PCR ZTargetl PCR 20 ul USI Target2 Referencel Reference2 PCR 20 7 PCR Ear aa a a X dvVh2EXNCUZERLLADPEBEEREBAVLTRUZL E E SUOMSUES 1 ng RNA Target 7 Lus o PCR 15 ul LightCycler 8 Tube Strips clear
28. Samples Samples 7 23 NTC No Template Control gt DNA StdDNA TET 7 2 1E3 1E4 Unkn A Unkn Color Name StdDNA 1E1 StdDNA 1E2 StdDNA 1E3 StdDNA 1E4 Unkn A Unkn B 23 GAN OREELU YS c EZBER P NIC gt Wells as Table Samples as Plate A1 B1 C1 D1 P Se Data E Samples Color Name Dye Reference de StdDNA 1E1 StdDNA 1E2 StdDNA 1E3 StdDNA 1E4 Unkh A 9 Set Number All Clear E Std y Unk l R Neg Clear EEN seesssme Targets as Piate 00000000000000000000000000000000000
29. DEEZER S Experiment 5 Run Settings M Profile Samples Temperature Data Intensity versus Time Intensity versus Acquisition Intensity 2 2 3 5 a E 2 1000 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 Acauisition Index Time minutes seconds VJ Correct for background Selected Channels Wells as Plate m 510 528nm 2 Note Sample Saturated 2 530 548nm 3 550 568nm 4 570 588nm 5 580 608nm 6 510 528nm 7 630 648nm 8 650 668nm 8 670 5688nm 10 680 08nm 11 710 728nm B2 10 Sample1 12 730 748nm s eme ResoLight Selected Channels 530 548 nm Wells as List Wells as Plate gt Correct for background gt LightCycler Nano Instrument F Version 1 0 High Hesolution Melting KEM 77 i ORE 4 3
30. F Version 1 0 ZEBLU FOE HN LightCycler Nano gt 16 24V DC 6 25A 16 Vil Q sonas sgomnemtihe censes LightCycler Nano Instrument LightCycler Nano Instrument LightCycler Nano Instrument LightCycler Nano Instrument A
31. gt gt gt SSS SS SS SS SS aS ae ELI ee ee ae ee ee eee ea o L ee eee ee ee eee MENENENENENENEN a 5 i A8 B8 C8 D8 li at Q AIIUSN TU SLEEMEEBL COSS S SESIZBIUTU LUPUS OPM ID RES SEND HUET 96 MWP 17 12 MWP 3 000 x g 30 LightCycler Nano Instrument F Version 1 0 X 4 4h2PEIXCUZERLILAPEEXCUtTBerUZL s tSUNUWES XERDZUDHZZILeZUCX T 3 2 Q ERDEI IY TEI OL EORR OTILE N VO FIROFUZFISEZEEF FT lt 3 2 1 18 SEESOERC DIRE C BARCEL 0 Run Settings Optics Settings 7
32. A ele2 Experiment Run Settings Ui Profile Data Samples Analysis Name Note Color Name Dye Reference Allele1 FAM Ju Sample 2 Sample 3 Sample 4 Sample 5 ES Samnie fi Wells as Table Samples as Plate Targets as Plate Pos 4 Note Sample FAM Type vic Type Al 1 ple u Allele2 u A A2 2 u Allele2 uj A3 3 uj Allele2 uj AA 4 ul Allele2 u A5 5 u Allele2 u AB B u Allele2 uj AT i u Allele2 u AB 8 u Allele2 u B8 D8 eg Wells as Table 27eETargets as Plate Wells as Table SamplesasPlate ERMETE SOEC 1 2 3 4 5 6 7 8 Target Assignments Select a target above to display whether it is an unknown standard negative or empty in each well If multiple targets are selected the first one is displayed B Key Bl Unknown C 39 Standard I Negative C
33. sample Cd values Cl by Sample v Dye 530 v Show all dyes LightCycler Nano Instrument F Version 1 0 PCROZEIPASYILE LEGS ING CHEM ES hae OBE DT 1 4 4 ays7js Experiment 3 Run Settings UL Profile Data Samples Select Analysis Analysis Stages Absolute Quantification Relative Quantification Auto Quant y Complete Intensity RFU 2 5 5 7 5 Loaxo q 5 10 15 20 25 30 35 40 45 Cycle Color by Sample vj Dye 530 v L Show all dyes Cq 3 3 logie q 37 24 770 9994 Error 0 225 E 2 009 Int Exclude Standards B Import 3 Export CeCe cari SamplesasPlate CqValues as Plate Pos Note Excl Sample 530 Type Cd Quantity 17 12 Bi Exon 477 7 97 7 results Analysis Stages Absolute Quantification Relative Qua
34. Y DEVICES Lj Macintosh HD H iDisk Remote Disc Y SHARED E itis s remot Y PLACES amp _ Applications MEL o Kd Desktop GS trepidacious A Applications Downloads gt dock dock gt Spotlight nano Nano Applications g MO S C 100 Tue 11 27 ey Show All Top Hit gi Nano Applications Definition noun short for nanotechnol LightCycler Nano Software dock Ag7o VAF LAOH DD LightCycler Nano Instrument N Version 1 0 LightCycler Nano System b PCROST TS 2L VES BNF EROS ER 16 b FONOREHMIRE Ty Calling H e EE 3B5N o gt QBRIEVUT TL OK QFEFACA ORE rT b EGO ER OTN Y gt High Resolution Melting 609 Pet SIAS A Cer 899
35. F Version 1 0 High Hesolution Melting da ORE 4 4 4na ysjs lt Run Settings Ui Profile Data Select Analysis Raw Settings Normalization Settings Temp Shift Settings Raw Normalized Temp Shifted Difference High Res Melt Apply Temperature Shift Color by Sample v Results as Table Samples as Plate Genotypes as Plate Pos Note Excl Sample Targeti Baseline Genotype Color Name Note Sample1 U UI UI a ri Assign UI UI Clear UI UI UI Bi Exon GQ results Normalization Settings Temp Shift Settings Normalized Temp Shifted Difference Target Target Zz Noise Reduction Range C D 1 re Increase range to reduce noise decrease to improve resolution Color by Sample Results as Table Samples as Plate Genotypes as Plate Note Excl Sample Target Baseline Genotype Sample1 Sample
36. LightCycler Nano Insttument ERS TUT A EM PCHOSTAZIiwZLLESHIBPCTSOfuLe FROP AIFS TERI 1 2 5 LightCycler Nano LightCycler Nano Instrument CN Show lag Phase 2 Cycling Phase cycle 11 stage 2 Stage Started About 28 mins left E Data Intensity versus Time Intensity versus Acquisition Please select dye s and samples below Temperature C 0 6 07 0 8 0 9 1 Acquisition Time minutes seconds Time minutes seconds V Correctfor background Selected Channels Wells as Plate 1 510 528nm Note Sample Saturated 2 630 548nm ki 3 550 568nm LightCycler Nano Instrument F Version 1 0 PCROZIPF SY PLE LES ING
37. SYBR Green I PCR Tn Tm Calling Tm Calling QO Analysis FERZEN 08 90 SDM ADORE ORET BIRR e Select Analysis gt Select Analysis Type gt Tm Calling Se ec7 Wy Automatic Quantification Absolute and relative quantification using automatic CQ calling Fit Point Quantification Absolute and relative quantification using fit point CQ calling m Tm Calling Melt temperature analysis Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping Spectra View Display of full spectrum optical data p en cae Se jngs Ana ysis Settings Melt Peaks Target Target2 V Use Negative Derivative Noise Reduction Ra
38. E EZI A oss lq F A OPE HERE Tm Calling EA AXES FROFOIF2 2 7 E fF 2 2 4 o LightCycler Nano Instrument 28 SOFT OIBE C S Bae Wila Q 97g7 Run Show log Data gt 29 Dala 7 2 27027277C 2c08FflIL 2v TIE LightCycler Nano Instrumet UIZ OSceN v Experiment 3x Run Settings Ui Profile Samples Analysis Temperature Data Intensity versus Time Intensity versus Acquisition 1000 90 HI WE MI MI im g 8 g 704 2 j Eco 50 l 40 30 8 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 01 15 00 Acquisition Time minutes seconds VJ Correct for background 0 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 Time minutes seconds Selected Channels Wells as Plate 1 510 528nm ja Pos Note Sample Saturate
39. 18 Run Settings Optics Settings SYBR Green Intercalating Dyes 24 L 7 4 7 E amp 3E c RARU amp d 40 A UO0 2 47 Of EERIK cC gt Normal Quality Please select the brightest dye present in any well If you are unsure use the Intercalating Dyes option Intercalating Dyes S Y BR Green ResoLight a Hydrolysis Probes FAM VIC HEX Yellow 555 Red 610 TexasRed Cy5 Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality lt L High Speed The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity Ifthe option is not selected the same settings selected above will be used throughout the run Optimize Melt Acquisitions ERS TUT A a E 7 KDEE EBHI RC Tm Calling EA AXES XBOZB
40. DNA 1 x10 1x10 10 LightCycler Nano Instrument F Version 1 0 PCHOZTZAZivZLLEP BSPCTOfES PCR 15 ul LightCycler 8 Tube Strips clear O 5 l 3 EE RSEN ERE ERES DICE SEES REEL cre o pp mE ee mc De oe MENECONNCMO e cse ode MENEENENNEN o o e so wD BEIFAALSENWSCEEHBLEC E amp USSZESICEIUCUERU RE TPOBICHEDPI amp 16124 Q 96 MWP MWP 3 000 x g 30 m uH PCHOSTAZIiwZLLESHIBPLTfums XBRDZDZTIiLeZCGAfr 1 2 gt gt LightCycler Nano I
41. Negative Targets Name Note Color Reference E Std V Unk Q A Clear Note Sample FAM Type oo 9 Set 9 Number All Clear Samples as Plate Targets as Plate m 2 D Q Wells as Table 7 Targets as Plate 1 2 4 5 6 7 8 Target Assignments Select a target above to display whether it A is an unknown standard negative or empty in each well If multiple targets are selected the first one is displayed B Key Bl Unknown 39 Standard Il Negative C Empty experiment bar Sgre Sagre Open Experiments TC 22 Dynamic Range HP 17 New 5 Save S Save As P Save 4s Save 45 Open Experiments TC 22 Dynamic Range HP 17 bd New E Save S Save As
42. 69 4 2 T1 4 2 1 A T1 4 2 2 TRO 72 4 2 3 kk 73 4 2 4 ee 76 4 3 FE ei co RR NR RR RETE 77 4 4 Lr d 81 5 ee 85 5 1 TO 85 5 2 mM H 87 5 2 1 De 05 p PT EE E NENEA E A EE S A E EE 87 5 2 2 FF 015 EENE EETA AA E TENE A AE AA E AES 88 5 2 4 DECUS Ac AO c NC e 89 5 2 4 OL 0 22 6 92 5 3 FE D ee compe RR 93 5 4 EA 95 C 97 D 99 1 TF A 99 LightCycler Nano Instrument F Version 1 0 BE Copyright 2011 Roche Diagnostics GmbH All rights reserved
43. EPF ice ar ica Samples asPlate Results as Plate Pos Note Excl Sample Call EPF 1 EPF 2 Al 1 Sample 1 7 96 231 468 a A2 2 Sample 2 Allele 1 94 558 1 544 A3 3 Heterozygous 58 49 136 535 Ad 4 ME 7 881 239 431 AS 5 A Allele 1 90 883 2 16 AB 5 Sample 6 Heterozygous 59 665 146 813 AT if Sample 7 9 832 251 359 AS 8 Li Allele 1 99 275 1 239 B1 g Sample 9 Heterozygous 71 404 178 121 B2 10 Sample 10 Alee 0000000000 7 258 235 735 B3 11 Allele 1 94 259 2 96 v B Export Results as Plate Results as Plate Resu ts as Table 1 Results as Table Samples as Plate Results as Plate 1 4 4 5 6 7 8 Genotypes Q Each well is colored according to genotype Sample Sample 3 Kev Gi Allele 1 Genotype Heterozygous Bl Allele 2 E Heterozygous C oS Q B No Amplification C Empty 490000000 LightCycler Nano Instrument F Version 1 0
44. FAM VIC os s Probes ANAK ART OT 62 gt Normal Quality Please selectthe brightest dye present in any well If you are unsure use the Intercalating Dyes option Intercalating Dyes SY8R Green ResoLight Hydrolysis Probes FAM VIC HEX Yellow 555 Red 610 TexasRed Cy5 i Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality mK The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity If the option is not selected the same settings selected above will be used throughout the run Optimize Melt Acquisitions Use Advanced Settings _ Display Dye Calibrations ERS TUT A X RDUDZLTSeZUCEfT 5 2 2 Program Temp C Ramp C s Ho
45. P Results74F 71U7 C Samples Targets amp 7 I amp Analysis Results ERS TUT A E X 4 hABEIXCUZERLILAPEXCHUtTBeUZL s tSUONBWES aR OREH Samples Samples Le ee anaysisresuns Name Note Calibrator heart A kidney La liver MZ H20 ddr Graph selected samples only Targets 7grgefs Samples Analysis Results Color Name Dye ES Targeti FAM EN Target2 FAM Reference FAM E Reference2 FAM Graph selected targets only Analysis Results Analysis Results 1 Samples Targets Analysis Results Sample Target IECorr Cq ACq Ratio Ratio Min Ratio Max A Cq Norm Ratio N R Min NLR Max 30 16 0 4 86 0 0 034 0 034 0 05 0 1 1 032 0 965 30 6 0 03 53 0 03 0 025 0 026 0 9 0 08 0 537 0 509 24 85 0 25 75 0 29 94 0 16 4 39 0 16 0 51 0 18 kidney Target 29 5 0 01 3 95 0 02 0 45 0 08 26 26 0 01 24 86 0 01 30 88 0 09 491 0 1 0 0 14 30 388 0 07
46. Results as Table Samples as Plate Cq Values as Plate 1 2 d 4 5 6 7 8 Display Settings Q Selected Dye FAM vj Range Minimum 0 Ej Range Maximum 45 V Use Automatic Range Each well is colored by its Cq forthe selected dye The same Cd range and colors are used for amplification 49000090 i E Cq within range C Empty Negative S8 Cq too early Wl ci too late LightCycler Nano Instrument Version 1 0 Bl 7 FKDEE EHI E Tm Calling EA AXES LD rk NO Tn Calling Cq Cq DNA PCR 1 PCR
47. Cq Cq Cq 2 2 1x10 1x10 2 2 x T Calliing NO i 2 PCR 1 2 20 ut BOE 14 Q LRP YT OP EF 2 71D EATER PT VEPCR 392 ZEIGE AET CUR io O JJ A amp NY N 1x10 1x10 ERS TUT A lq F AOE HERE Tm Calling EA
48. gt FAM Color Name SYBR Greer TexasRed v Wells as Table Targets as Plate A1 A3 cap S 1 Oo OO Name Color Name Reference S 100000000 S 100000000 S 100000000 Input m Cn ECL m CO 38 2 1 000 000 000 C652 amp 1E9 El 1000000000 OLED U SADEA TILE GF E55 ORC COE La TIE d W Please input standard quantity must be a number greater than 0 1E9 Cancel 5 8 A4 D6 gt gt gt LightCycler Nano Instrument Version 1 0 PCHOSTAZIiwZLLEEHSIBbCTI XLES XERDUDHZZILZUCX T Q D7 D8 Meg
49. 25 Normal Quality ZE Ig ET e AR UC JESE 3 ORG S EE SE U s Please selectthe brightest dye present in any well If you are unsure use the Intercalating Dyes option Intercalating Dyes S Y amp R Green i ResoLight Hydrolysis Probes FAM WIC HEX Yellow 555 Red 610 TexasRed Cy5 s Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality S The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity Ifthe option is not selected the same settings selected above will be used throughoutthe run Optimize Melt Acquisitions Use Advanced Settings _ Display Dye Calibrations PCROZIP SY PLE LESIONS SEXES EFROP AIFS TERI 1 2 3 Program Temp C Ramp C s Hold s Profile
50. Negative S dM A8 B8 C8 D8 Standard wv wv Targets 4 RITI 8 4 25 4 7r v OBE EIU 4 FAM gt Referencel amp Heference20 Reference Targets Color Name Dye Reference dp Target 2 FAM Au Ld Reference1 FAM AF E Reference FAM A P Target gt Wells as Table Samples as Plate 47 gt 46 Unk P Target gt O 4 Z
51. 32 LightCycler 8 Tube Strips cleary 4 b BERE TVMOS E PPCROZUuZZ2AEbZ270H ih b gt 11 LightCycler Nano Instrument DER Diz CBR 4 738 vs gt 12 LightCycler Nano Software LightCycler Nano Software Z 774 WV2L V 4 vs 7x X03 x0g amp Bs OAT ii LightCycler Nano Instrument N b 4 X 9 FIO 7 hvxrjo cA ZE E e oo POR DLL I SY PLE SES D ITEXT EE 1 PCR 1 ml
52. Data TOT FIERO FFM OUTIL LightCycler Nano 775 77797 OS CCSBHB GeV Experiment Im Temperature Data Intensity versus Time Intensity versus Acquisition 8 Temperature N eo 16 40 25 00 33 20 41 40 50 00 5820 01 06 40 01 15 00 0 16 40 33 20 50 00 01 06 40 Acquisition Time minutes seconds Time minutes seconds VJ Correct for background Selected Channels 1 510 528nm A Pos E Note Sample Saturated 3 550 568nm 4 570 588nm 5 590 608nm B 610 628nm 7 630 648nm 8 650 668nm 9 670 688nm 10 690 708nm 44 740 7 0 B 1n amp amnla 1f D v Selected Channels gt FAM 239 548 nm VIC 550 560 nm Wells as List Wells as Plate gt Correct for background LightCycl
53. Export Q T R FO Ctrlle a t amp f amp Results as Table Z0 3 CO V TF WEERLEF gt gt gt EZI A High Hesolution Melting de OREN 2 gt Genotypes p gt Color Name Note difference plot curve eye 7ce gt Raw Normalized Temp Shifted E OO Notes RFU 73 74 75 76 77 78 Temperature C Color by Sample v gt Assjgn ek cis Normalization Settings Temp Shift Settings Target Target v Noise R
54. ResultsasTable as Table Samples as Plate Tm as Plate Note Excl Sample Target2 NTC StdDNA 1E1 u u D1 25 NES D2 26 gtdDNA 1E1 iSi 10 72 68 E Bi Export Melt Peaks Results as Table z amp CHARE Melt Peaks EI EU 7 1 Melt Curve Notes 60 65 70 75 80 85 90 95 Temperature C Tm as Plate Tm as Plate 7 L Gh A Tg f Peak Area for Plate Display 7 LL hzzs OE Z B185 NEZKA SIAI KELET Use Peak Area Range lc quU Ad Ed Results as Table Samples as Plate Peak Area for Plate Display Selected Peak Area Peak 1 65 45 to 81 5 x lt Tm display range Range Minimum 50 Ej Range Maximum j 100 P V Use Peak Area Range a Each well is calored by its Tm for the selected peak area ES Tm within range C Empty Negative BH Tm below minimum SS Tm above maximum LightCycler Nano Instrument
55. Version 1 0 2 9 b 2IERAEY DP LER 2FERAE M IEF FU TL 8 EBOCHIER 3 2 2 2 F ratio ratio ratio 1 ratio ratio 1 2 2 2
56. gt No of Cycles 45 Column Value Step 1 Value Step 2 Name 2 Step Amplification Hold 95 C for 600s No of Cycles 45 EJ Temp C Ramp Cis Hold s Acquire on m OMNE 45 cycles of 95 C to 60 C 95 5 20 zc add Delete Up 3 Down add Delete up down Q F ORBINA OBIT OT SL EES ELBZILBVE GA BA ORE EH LU ZISESIBSI 0 C IAE T gt gt gt SU I Eu PCROZEIAS 9 PLE LEGS ING SINTER EROFOAI ASS ZERIT Temperature Profile Temperature Profile Time minutes seconds Name 2 Step Amplification Hold 95 C for 600s No of Cycles 45 S Temp CCQ Ramp Cis Hold s Acquire on EE cycles MM dada Delete J MUp oon Add Delete Up 3 Down LightCycler Nano Instrument F Version 1 0 PCROZIPF SY PLE LES ING EIEE XERDZUDHZZIDLZUCX T 1 2 4
57. Q SE HEIL XN TOVL TN EEN By 4 TZA Number AIL E EBEEREUE T Wells as Table Samples as Plate Wells as Table Samples as Plate Targets as Plate 5 6 7 8 Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray E ORO a TD 27 XE OZDZZieZC Efr Targets 2 25 v OE CEI 4 e e7 4 e e2 gt 4 g e7 FAM dye 4A ele2 VIC dye Color Name Dye Reference db S EE Allele1 FAM ES gt Allele1 gt Wells as Table 2 Targets as Plate B8 D8 Unk
58. LightCycler Nano Instrtument LightCycler Nano Instrument ow w V gt BY BRK B k ee gt LightCycler Nano Instrument LightCycler Nano Instrument
59. Results as Table as Table Samples as Plate Cq Values as Plate Note Excl Sample 530 Type CH Quantity A1 1 xus 1000000000 723 A 1 228 910 689 848 A2 2 S 1000000000 7 236 A 1 224 110 985 678 A3 a 1000000000 7 298 A 1 172 273 508 519 Ad 4 S 100000000 10 712 A 108 283 882 885 AS 5 S 100000000 10 774 A 103 740 399 3 Ab B is 100000000 10 789 se 102 647 910 597 Af 7 10000000 14 183 A 9 622 081 407 A8 8 S 10000000 1418 A 9 642 823 056 B1 g S 10000000 14451 A 9 838 900 197 B2 10 S 1000000 17 541 A 924 733 857 E D3 11_ ET rt MNA 1nnnnnn 17607 a n474100547 Lv Ba Export Samples as Plate Samples as Plate CZL h CHE VOT I Results as Table Samples as Plate Cq Values as Plate 1 2 3 4 5 6 7 8 Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray Cq Values as Plate Co Values as Plate CZ lL F cfi ACHE Cq Display Settings Use Automatic Range
60. Wells as Table Samples as Plate Al1 A2 A3 gt gt gt LightCycler Nano Instrument Version 1 0 PCHOSTAZAIwZLLEEHSIBbCTI X ES XERDZUDHZZILZUCX T Q Se7 Name Note Color Name Dye Reference db e Aca 9 Set 9 Number All Clear E Std v Unk inj Neg Clear ES AEI SamplesasPlate Targets as Plate Pos Note Sample Q 7 9 Wells as Table Samples as Plate Wells as Table Samples as Plate Targets as Plate 1 2 3 4 5 6 7 8 Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray
61. i Profile Temperature Profile Temperature C e Programs 4 AK Ada Choose Predefined Programs Add Delete A Up A Down e Hold MBA POTS LEER Select CEIR LIZ 45 cycles of 95 C to 60 C VA 3 Step Touchdown T D Amplification 45 cycles of 95 C to 65 C to 72 C a uum 60 C to 95 C at 0 1 C s UU High Resolution Melting 60 C to 95 C at 0 05 C s Cancel og7g77s Temperature Profile gt gt gt LightCycler Nano Instrument Version 1 0 PCROZIPF SY PLE LEASING EIEE XERDZHZZIiLZCE T Hold gt Name gt Tempo C 95 gt Ramo C 2 4 gt Hola s 600 Name Hold Hold 95 C for 600s Temp C 95 n Ramp C s 5 Ej Hold s 600 Delete Up A Down Q 2 Step Amplification Cyc ing gt Name
62. Applera Corporation IV FA LightCycler Nano Instrument DNA PCR LightCycle Nano System LightCycler Nano Instrument LightCycler Nano System V FX LightCycler Nano Instrument VI
63. Mm Radius NA Cycles Min Angle 30 p Max Angle 75 772 is Cyc es Targets Thresholds First Background Cycle 4 EJ Number of Background Cycles 3 Ej amp Number of Endpoint Cycles 3 i LightCycler Nano Instrument F Version 1 0 7 ia ORE 5 4 Analysis Experiment SRun Settings Ui Profile Data Samples Analysis Select Analysis Targets Thresholds Cycles aA Endpoint Allele 1 Allele1 M Allele 2 Allele2 v Complete OSACE SamplesasPlate Results as Plate Pos Note Excl Sample Call EPF 4 EPF 2 1 4 i a Export e Endooint Graph
64. Results as Table Samples Analysis Stages Absolute Quantification Quantifiers Amplification Selected Target Targett v Amplification Notes Excluded early cycles 3 iid 5 0 75 Min Relative Amp 0 1 iS gt 05 2 Min Amp Quality sill 0 25 5 10 15 20 25 30 35 40 45 Cycle Color by Sample M Dye SYBR Green v Show all dyes Results as Table Samples as Plate Cq Values as Plate Pos Note Excl Sample SYBR Green Type Cq Quantity A1 1 c BB Target A A2 2 SItHDNA 1E1 Target E 10 32 091 A 10 001 A3 3 A Target1 5 100 28 86 A 91 873 Ad 4 Li Target1 1000 25 331 A 1 034 984 AS 5 hee Target E 10000 21 904 A 10 870 556 AG 6 Unkn A Target1 uj 26 432 A 486 243 AT 7 1 UnknB Target1 u 33 845 A 3 002 Ag 8 B1 g U BE Targeti em gt gt gt EZIO AAE RA Emu Bl 7 KDEE EBHI RC Tm Calling EA AXES BV E S amp Automatic Quantification Settings Quantifiers Amplification Selected Target Target v uM Excluded early cycles 3 p Min Relative Amp 0 1 Min Amp Quality s ej
65. Pe target Cy5 Q ARVE QBZBZ2J Z444 c c Target 1 h5 Targe txl CO BIOS 7 yh EBD GROANHEI CAF 75 Color Name Dye Reference db e Co o Select color OX gt gt gt EZI A E PCHOSTAZIiwZLLESHIBPITSIfumE FROP AIFS ERIT Targets Color Name Dye Reference IG O Dye
66. Quantifiers E 2 1 2 Q EUEBIARO SERI YE SER AR ET XA HED AED CREAN SLUG SET Settings Amplification Selected Target Target v Lod1o q Cq 3 36 logie q 35 45 770 9979 Error 0 17 E 1 986 Int Exclude Standards Import Export Intensity RFU Color by Sample vj Dye SYBR Green vj _ Show all dyes LightCycler Nano Instrument F Version 1 0 Bl 7 KDEE EHI RC Tm Calling EA AXES BAEZ Automatic Quantification 2 3 2 4nalysis 3 Run Settings UL Profile Data Samples Analysis Select Analysis Analysis Stages Absolute Quantification Relative Quantification Quantifiers Amplification Selected Target Target Excluded early cycles 3 n Min Relative Amp 0 1 n Min Amp Quality 5 lii
67. Group2 O co N cC A A ct N oOo lu lu lu lu lu lu lu lu lu lu lu Export SU I eA EN High Hesolution Melting de OREN Genotypes as Plate Genotypes as Plate Genotypes 81 FROM 3 Results as Table Samples as Plate Genotypes as Plate 1 2 3 4 5 6 7 8 HRM Genotypes Each well has the same color as the genotype assigned to it Wells with no sample are gray 009000000 00000000 LightCycler Nano Instrument F Version 1 0 NICO Ae LAR CA7 5 DNA 2 2 PCR
68. gt Neg gt gt gt LightCycler Nano Instrument F Version 1 0 Fy h QIEREV IP LER 2AA ERr UZLL iib EE XERDUDHZZILZUCX T 4 Target gt A8 SS 077 25020 Targets Color Name Note Color Name Reference Targett it ES kidney ag liver Reference H20 n Reference2 Note Sample FAM Type Q 2 4 58 Target2 Reference Reference2 Wells as Table Targets as Plate Wells as Table Samples as Plate Targets as Plate Target Assignments Select a target above to display whether it is an unknown standard negative or empty in each well If multiple targets are se
69. 7 Auto Quant Complete Settings Notes 0 75 0 5 Intensity RFU 0 25 5 10 15 20 25 30 35 40 45 Cycle Color by Sample jv Dye SYBR Green M Show all dyes Results as Table Samples as Plate Cq Values as Plate Pos Note Excl Sample SYBR Green Type Cq Quantity StdDNA 1E1 10 100 1000 10000 21 904 Export e 477 7 cg 7 results Analysis Stages USOT fe oT Quantifiers Amplification Selected Target Target1 v Excluded early cycles 3 EJ Settings Amplification N m Min Relative Amp 0 1 EJ Min Amp Quality 5 5 Intensity RFU 10 15 20 25 30 35 40 45 Cvcle Color by Sample Dye SYBR Green Di Show all dyes Toner Samples as Plate CqValues as Plate Note Excl Sample SYBR Green Quantity StdDNA 1E1 10 001 100 86 91 873 1000 10000 Q C J al Results as 79 6 gt gt gt EZI A Bl 7 KDEE EBHI RC Tm Calling EA AXES BEI E Automatic Quantification e
70. Noise Reduction Range C Increase range to reduce noise decrease to improve resolution 80 85 Temperature C Color by Sample v Results as Table Samples as Plate Genotypes as Plate Bi Export Pos Note Excl Sample Target Baseline Genotype Color Name Note a ll lt A3 3 Sample3 uj m A4 4 Samplet u AG B Sample3 u Clear A 7 el lu AO o Ii gt gt gt EZI A Eno High Hesolution Melting BEMI iO BE P Results as 720 6 gt Excl Results as Table Samples as Plate Genotypes as Plate Pos E Note Excl Sample Target Baseline Genotype D2 26 uj D3 27 5 Sample3 u D4 28 Samplet u D5 29 a uj De 30 ZY Sample3 uj D7 31 ee u D8 32 Y Neo j Raw Settings gt gt Noise Reduction 7g77ge Normalization Settings Temp Shift Settings Target Target v E Noise Reduction Range C 0 1 Increase range to re
71. Notes 10 fold dilution series from 1E9 to 1E0 copies with FAM labeled hydrolysis probes LightCycler Nano Instrument F Version 1 0 PCHOSTAZIwZLLEEHSIBPCTI XES XERDUDHZZIDLZUCX T 1 22 e Run Settings Optics Settings A Run Settings fu Profile Samples Please selectthe brightest dye presentin any well If you are unsure use the Intercalating Dyes option Intercalating Dyes SYBR Green ResoLight J Hydrolysis Probes FAM VIC HEX Yellow 555 Red 610 TexasRed Cy5 Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality High Speed The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity Ifthe option is not selected the same settings selected above will be used throughout the run Optimize Melt Acquisitions FAM Hydrolysis Probes
72. Number of Background Cycles 4 ij 2 Number of Fit Points 2 Ej E Thresholds VJ Automatic Quantification Fold Global Noise 20 bad 5 10 15 20 25 30 35 40 45 Cycle PosJNeg Fold Global Noise 100 m Color by Sample Dye SYBR Green v J Show all dyes Quantity M 1 Target1 U A2 2 Target ISl 10 30 329 A 10 012 A3 3 Target IS 100 27364 A 78 619 A4 4 Target IS 1000 23 559 A 1 107 842 A5 5 Target S 10000 20 376 A 10 122 419 AB 6 i UnknA Target u 24 754 A 482 63 A 7 i UnknB Target u 31 998 A 3 138 AB 8 B1 g sE Target gt gt gt LightCycler Nano Instrument Version 1 0 Bl 7 KDEE EH RC Tm Calling EA AXES Fit Point Fit point Quantification Settings I Quantifiers Amplification Selected Target Target2 v 85 First Background Cycle 2 EJ Number of Background Cycles 4 n Number of Fit Points 2 n V Automatic Quantification xFold Global Noise 4 n PosJ Neg xFold Global Noise 25 EJ Cycle Settings Thresholds Cycle Settings gt First Background Cycle gt Numbe
73. P Set Name Color Name Dye Reference E Sample2 Sample3 NTC wells as Table Samples as Plate Targets as Plate Note Sample gt gt gt SU I eA Hou High Hesolution Melting XERDZHZZSLZCEfr Q 70 Wells as Table amp Samples as Plate Wells as Table Samples as Plate wensas Table MEER Targets aspie 0 as Plate Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray amp b Targets gt ResoLight 25s Z0 2 4 Kh OMEEEI VY Color Name Dye Reference ResoLight Target d Wells as Table Targets as
74. 44 0 07 0 0 11 Bi Export LightCycler Nano Instrument F Version 1 0 High Hesolution Melting 4 High Resolution Melting High Resolution Melting PCR DNA DNA 2 dsDNA 1 ssDNA High Resolution Melting dsDNA DNA ResoLight ssDNA dsDNA PCR DNA DNA High Resolution Melting SNP
75. AXES DE y hPyT e Target1 PCR 15 Target2 PCR 15 ul amp LightCycler 8 Tube Strips clear gt gt gt ES SS SS aS eS LESS ee Sr EIE ei E RON DERSEC NN 0 ee ee e ds ee a ee ee ee ee ee e 5 ul Standard 5 ul unknown IRID Q Sas BILSH TL SCEEMBEBL COR SEASCBEU TU GAHULS OPM PED RET SEP DUET BERD ST SEMI Tai 2ARUJZ 1EE096XMWPIZREXIBUES 17N YO zb DIRE C S RR Uv MWP 3 000 x g 30 LightCycler Nano Instrument F Version 1 0 E 7 KDEE EBHI RC Tm Calling EA AXES XEBOZHZTSLZCXE T 2 2 ERDE IY FEFITOR VEO RAN IOW CIE ISN YL Ol OX cH 0 1 2 2 1
76. Peak Area Temp 1 Temp 2 Threshold dF dT Delete Up A Down 90 95 Temperature C Melt Peaks Tn Q NA ATK ILO Sad FMENSE IRAE ERROEL UCASE V ATK AO m ER ORR X NEE Ys Settings Peak Areas Target Target v Peak Area Temp 1 Temp 2 Threshold dFidT db Add Delete Up 3 Down 90 95 Temperature C LightCycler Nano Instrument Version 1 0 lq F ADEE HERE Tm Calling EA AXES Tm Calling 2 5 2 4 g ys7s Select Analysis Settings Peak Areas Experiment Samples Analysis Melt Curve Notes Wy Auto Quant Target Target2 v Complete p WV Use Negative Derivative z Fit Point Quant Complete Noise Reduction Range C 1 n Jn Tm Calling W Complete Increase range to reduce noise decrease to improve resolution Results as Table Samples as Plate Tm as Plate Pos Note Excl Sample Ta
77. SYBR Green l Note Sample StdDNA 1E1 CO s Cc UC A N LL oO B1 B2 10 StdDNA 1E1 ISl 10 v gt gt gt LightCycler Nano Instrument Version 1 0 lq F AOPEHEIRE Tm Calling EA AXES FROFVOIF2 7EX i 36 2 4 7arget2 2 Wells as Table Targets as Plate Wells as Table SamplesasPlate ETT CE FC 1 2 3 4 5 6 7 8 Target Assignments Select a target above to display whether it is an unknown standard negative or empty in each well If multiple targets are selected the first one is displayed Key Bl Unknown 99 Standard Il Negative C Empty e experiment bar Save Sgre P Save 4s Save 4s 27 N Z00 ERRO
78. TI Results SNE TIA 83 R n Settings 7 7 ai ee 71 omes C ea S fi Temp Shift Settings seneesa 79 a T T 82 ET OCI NII TN cl 69 Lr 73 hp o E 74 A 76 RM ccc 7 L S T fi DI EE 7 LightCycler Nano Instrument zc 9 8 E m 11 We 97 E E il NT H 9 LightCycler Nano Mac OS X Lee 13 Microsoft Windows 12 ai 97 M Melt Curve dona EE i i Ue tit D 54 Melt GUT OS crista decent tcu da eee tice rA b UR RR 54 Melt Peaks A 54 Microsoft Windows LightCycl r Nano SoftWare assesses 12 IN easter tee eee fi Index N Normalization cua cecmeere EUR etr crt deep ec eb EE Rc 79 Normalization Settings high resolution melting lr 79 Ur E E E ANEA 7 P 7 MNO 16 POR 9994 high resolution melting ll 69 mos aq cR E UE Loire 85 0p MC i 16 35 A E E N AE AA B SANG 7 Pe 54 Peak Areas Duda eese uM E EL D 54 Pro S d an 20 38 60 72 88 Q 4 2 0H NR A NOR 7 Quantifiers Fit Point Fit point Quantification usss 50 i cra 32 44 iP x RR 66 H Raw Settings high resolution melting ss 71 78 Raw high resolution melting ee T1 Relative Quantiticatlon aucun 66 Results as Plate EA Ps OPP EP
79. amp fFfliI ov Vct LightCycler Nano 7 777977 OS amp cSWA Cav s Experiment 3 Run Settings V Profile Temperature Data Intensity versus Time Intensity versus Acquisition Temperature C Intensity 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 Acquisition Time minutes seconds 0 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 V Correct for background Time minutes seconds Selected Channels Wells as Plate 1 510 528nm Note Sample Saturated 2 530 548nm i 3 550 568nm 4 570 588nm 5 580 608nm 6 610 628nm 7 630 648nm 8 650 668nm 9 670 688nm 10 690 708nm 11 710 728nm FAM Selected Channels 530 548 nm Wells as List Wells as Plate gt Correct for CKg7O7779 LightCycler Nano Instrument F Version 1 0 X 4 4hAPBEIXCUZERLOILADPEZCHUtTBLeUZL s tS
80. are selected the first one is displayed B Key Bl Unknown E 39 Standard B Negative z C Empty experiment bar Sgre Sgre P Save 4s Sgve As 227 270 ZHAO ig ERS TUT A E High Hesolution Melting XERDZHZZSLZCEXfr 4 2 4 LightCycler Nano Insttument 28 CQ Start AM Show log ll Abort Run Data 29 Data 2IDTFTFERO FFHE OU CIEL LightCycler Nano Instrumet
81. dyes LightCycler Nano Instrument F Version 1 0 lq F A OPE HERE Tm Calling A AXES Fit Point Fit point Quantification 2 4 2 Analysis Cq ES Experiment Select Analysis i Absolute Quantification Relative Quantification g NE NE crews Quantifiers Amplification Complete it Poi 5 Selected Target Target v Fi Fit Point Quant g g Complete T Cycle Settings First Background Cycle 2 n Number of Background Cycles 4 E V Automatic Quantification xFold Global Noise 4 E Pos Neg Fold Global Noise 25 e Intensity RFU Thresholds COSAC SamplesasPlate CqValues as Plate Note Excl Sample SYBR Green Target2 StdDNA 1E1 Target2 10 Target2 100 Target2 1000 Target2 10000 Target2 Unkn B Target Target2 M StdDNA 1E1 Target2 A 7 522 Export J gt GD neu 97 7JV amp 338RU X d Amplification results Quantifiers Amplification Selected Target Target2 v First Background Cycle 2 EJ Number of Background Cycles 4 n Number of Fit Points 2 EJ W Automatic Quantificati
82. i i eee 96 Results as Table high resolution melting rr 83 Tat ni El DD AP as NAAA E i 96 ME TE 33 46 51 RE 7 A 28 42 64 76 92 pr i P 19 37 59 71 87 VU Nc 15 ure M 29 42 64 76 92 zd E 1 P 20 Rum Settinigs AP ARR RR RR 19 37 59 71 87 S vM ce JT 23 high resolution melting ll 19 a pg EO eee 89 QUO ce NR NN VIS 0 DIU ON cA A 61 66 Bu MM eee Settings Fit Point Fit point Quantification ss 49 LightCycler Nano Software s 97 Tr CA e Dd Automatic Quantification 32 44 LightCycler Nano Instrument 97 EE ee 7 oo 7 Targets nap ND 2 P 93 Temp Shift Settings high resolution melting Saute 79 Temperat ra SHI voee eibi ll 79 Thresholds POST Pa A ER 94 c decent eee mE TRUE 7 Tm as Plate iU iln 56 Tm calling PREVA SV 2 d 59 Melt CU ce ntu etai tanda 54 Melk Peake 54 Peak Arens TT a 54 PO d 38 Results as Table ee 05 elm d 99 PUE PeT T P 56 Ta Cime Mm Do Oa PLA n Em 35 U iW T T 7 V Aor mu T L E M 17 36 58 70 86 uad ll 85 93 LOL 220 A P E 95 ccr a de 94 D cR 02 Endpoint Crap UP cal
83. 00 Pos Note Sample A1 StdDNA 1E1 1 A2 A3 A4 A5 i Unkn A AT zT AB B1 i 36 Me s as Table Samples as Plate Wells as Table Samples as Plate Targets as Plate 1 2 3 4 5 6 7 8 Sample Assignments Each well has the same color as the sample assigned to it Wells with no sample are gray ERS TUT vary za lq F AOE HERE Tm Calling EA AXES FROFOIIF2 2 EZT Q 2gef5 2 25 SYBR Green Color Name Dye Reference Targett SYBR Green I E Target SYBR Green wall 1 M 9e gt Target 1 47 7 P Neg
84. 37 DIBG2 RNN KS S y 7T RT DDT b b 23 39 SUCTUS IST RU damcnapmitesangendunid 16 35 pror MmMr 23 39 Automatic Quantification 30 43 imd us P 25 40 etn ao a sea litium e ETE 29 42 ON P 20 QU i eee Se 11 WWE Index J 20 cas ae NA E MEAM MEM E EE 57 Analysis Besultg Luuuousueceii nemen 67 DONC RANT 67 EE EE 64 DCR coos etree retin ett ee 57 ly a 60 VAC OD osassadtetezantacdibd esatta en 66 R n settings F 7 enpe huie nerd 59 vcn m S NA 61 66 00 D Ur 61 Automatic Quantification s 65 2o 62 2c J 64 high resolution melting 74 NNA VE ap o E MP Le sme 90 Up IG MR RR RE 25 40 HU at RR 62 IB FO ee eos todatif atro bolt tide 35 JN vl 11 high resolution melting s T4 Tace t TETTE 38 APRA SPY SIPS COZ III 88 EE E cl 20 38 ALTUS ELE E 60 Published by Roche Diagnostics GmbH Sandhofer StraBe 116 68305 Mannheim Germany 2011 Roche Diagnostics All rights reserved www roche applied science com D 0611
85. 94 PERO UU M unneeidutrccu D EU EE 85 Ia voir E Br PR PT 88 Results as Plate FF snusare ora 96 kecute as Tibe a 96 R n Dette TT uaa a 87 Samples c V Qe 89 dicc cq 2c 93 Thresholds gee an ee ven EE en eT 94 IW agen ei EU BU a 93 TN cr 85 i217 T 89 LightCycler Nano Instrument Version 1 0 xA PIE 90 cu dean ele 92 LightCycler Nano Instrument ll Mac OS X LightCycler Nano Software 13 mau LP ro 66 high resolution melting ee 71 ERA GI ELTE UT a 87 i pcs A 19 37 emo 59 ELGG lA 7 high resolution TGM uunc ertet 69 Josue e cactus NMa iem EE GIC 9D RNN du dk 85 Sb APR 16 35 a E o EOR RS 57 c2 09 uae 21 5 63 15 91 Automatic Quantification s s 30 43 65 Amplincation 7 7 eese enemies 33 4b 5L 55 A S T raa 32 44 er asinum a M 32 44 t Ec 16 35 Na DA 99 45 DI Ca Values as Plate aa 34 47 52 lp UNS A 29 42 Fit Point Fit point Quantification 48 PERO AT VFA setate utei p 16 POR D 16 35 Pi e M 20 38 Results as Table sse 33 46 51 Pum ete So T ama OA Haa op 19
86. CRT 65 67 C Sg ree eee ea en OE aT nS Tm eT Ona TIE Cee RE aT ome PPI Te 7 Cq Values as Plate prole eda eet tend 20 38 60 72 88 18 i ds cuc c 34 47 52 O a RES EA IONE ASAA SAITA A 7 Cycles 77 LAA v JOUET etree 94 D UD Me 29 42 64 76 92 Difference Meh resolution Meng Lotti et 80 E D 7 Endpoint Graph d Rd rcd A 94 I T Experiment Summary ee 18 TE 18 ER ONT 28 42 64 76 92 c aes 27 41 63 75 91 peram 29 42 64 76 92 joe qe uiu ine d m 18 F ow M eters verte neat A 7 Fit Point Fit point Quantification cscs 48 Amplification F Lats ated n diari ER rd 50 QuantiflQES a 50 DA 49 G Genotypes as Plate high resolution melting ue 84 WWE Index Genotypes high resolution melting lr 82 EE M 9m 7 H iplo T T High resolution melting 69 77 Analysis rae m 8l I c a e 76 Difference 7 7 ARR IRR RR EE ER RR 80 Gernotypes as Plate 2 7 uu setae ten dedans 84 High Resolution Melt analysis LA uU Normalization Settings usse 79 PER rU 69 luco e ar T eee ee 72 ICA DOGS m a ee 77 78 TR a e tte
87. E E A E E E EE 33 2 Tm Calling kk 35 2 1 PEPA alc 35 22 FOF DF T Dra S a IEEE 37 2 2 cS 37 299 kk 38 2 2 H v 39 2 2 4 PEE 42 2 3 Automatic Quantification 43 2 3 1 eee 43 2 3 2 l0 ns 45 2 4 Fit Point Fit point QUANTIFICATION kk 48 2 4 1 ee 48 2 4 2 51 2 9 Ta 6l c 53 2 5 1 ae 53 2 5 2 X 55 3 2 2 oo 57 3 1 puts bci 0 E E EE EASE EEA E AE E A EE A A 57 3 2 kk kk 59 3 2 1 sic 59 3 9 9 as 60 3 2 3 AD IV T NU Tp WERE 61 3 2 4 E E A A 64 33 scesostie cede E AA AN AE AEE ENSE OAA AA A AASE 65 3 4 cs 67 AX 4 High Resolution Melting i 69 4 1 AD IV Lua A IMMME
88. EIEE XERDZUDHZZIDLZUCX T Data TOT FIERO FEW OV CIE LightCycler Nano 5 ge v ZH DEEZ BHC FAV Temperature Data Intensity versus Time Temperature C 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 01 15 00 01 06 40 Acquisition Time minutes seconds Time minutes seconds V Correct for background Selected Channels Wells as Plate 1 510 528nm Note Sample Saturated 2 530 548nmMm 3 550 558nm 4 570 588nm 5 580 608nm 5 610 528nm 7 630 648nm 8 650 668nm 9 670 688nm 10 690 708nm 11 710 728nm FAM Selected Channels 52 548 nm i TRU amp 3 Wells as List Wells as Plate gt Correct for background 7 v ZZ 22V FTBIE 4 7 aT ry7 A LightCycler Nano Inmstrument
89. EROP AIFS ERIT Co o Color Name Note qn Sample 2 ES Sample 4 Sample 5 Sample 5 Sample 7 Sample 8 Sample 8 d Sample 10 Y Sample 11 gt Select CoO Select color xi Color Palette OPPEREN B8 5 BES EESESESESIN ILS LI EEE Previous Selected Lo Cox aie QO 16 Samples Color Name Note TP ES 163 E 1E7 s E 1E5 E ES 1E4 i5 ES E 1E2 E 1E1 Ld 1E0 a E H20 O Aojes e 1E9
90. Empty LightCycler Nano Instrument Version 1 0 XBODZDUZZieZC fT e experiment bar gt Save Sgre P Save As Save 4g 277 270 ZO RI ERS TUT A E TAF TAN IAA X OZDZZieZc Efr 5 2 4 LightCycler Nano Instrument 28 Start Run Show log NI Abort Run Data 29
91. HZTSeZCEf r 2 2 2 Program Temp C Ramp C S Hold s Programs 20 Oo Profile e Temperature Profile Temperature Profile 100 z a 8 50 o B 25 o 0 0 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 01 15 00 Time minutes seconds Name Melting Hold Temp C eo f Se amplification 45 cycles of 96 C to 58 C to 72 C Ramp C s 4 n Hold s 60 Hold 60 C for 60s Final Stage Temp C 97 P Melting 60 C to 97 C at 0 1 C s Ramp Cis 0 1 p db Add Delete sup Dom LightCycler Nano Instrument F Version 1 0 Bl 7 KDEE EBI RC Tm Calling EA AXES XEBOZHZTSCZCXE T 2 2 3
92. Light4 Cycler LightCycler Nano System 1 0 For life science research only Not for use in diagnostic procedures AX l 5 NRRREERRRERERREEEI RE 5 Ii por ONMMEETEMOEEERPE evetassa 6 IV NNNONEENOO P9A 6 V v O tess 6 VI 6 VII 6 VIII 9 1 LightCycler amp Nano Instrument ae 11 2 LightCycler amp Nano Software te 12 B 15 1 PCR kk 16 1 1 AD IV I EM 16 1 2 OD FF ui TERRIER RUE 18 1 2 1 18 1 2 2 eo 19 1 2 3 rino 9 20 1 2 4 sU A LE 20s m c me S 23 1 2 5 oeccccccccceccccccssecucccssesucccssscsecssssucecesssaueccssaasecssauacesssauecessssusecsesusecesecauecsesasecessscuscessseaeeeessues 28 1 3 FEN I E AREE E EE AE E EEE AAEE ES 30 1 4 EAA E E E E
93. Plate B8 D8 gt Unk gt gt gt LightCycler Nano Instrument F Version 1 0 High Hesolution Melting EFROFOAIFS ZERIT B8 D8 P Neg Targets Color Name Note Color Name Reference Target 1 ResoLight m Sample2 Eni Sample3 E NTC Samples as Plate Targets as Plate Pos Note B5 13 Sample Sample C1 17 E 0 c c EM C e e s C2 18 Bample3 C3 18 Sarnplet C4 20 C5 21 CB 22 Samplet Yer 73 Wells as Table27 Targets as Plate Wells as Table Samples as Plate EMET ETOC 1 2 3 4 5 6 7 8 Target Assignments Select a target above to display whether it A is an unknown standard negative or empty in each well If multiple targets
94. Quantification Cq Pos Neg EC Quantifiers Amplification Notes Selected Target Target2 i v First Background Cycle Cycle Settings Number of Background Cycles Intensity RFU Number of Fit Points Thresholds CJ Automatic Quantification RFU 42 001 618 n Pos Neg RFU 109 065 433 Ej Color by Sample v Dye SYBR Green v _ Show all dyes Qug77 erS E 2 1 2 Amplification Selected Target Target2 35 32 5 30 g ce 275 25 22 5 Loa o q Cq 3 93 logie q 37 8 r 0 99 Error 0 441 E 1 797 Int Exclude Standards Import Export 7 Amplification 2 Tit HRO 68 7 V 2v C by sample by Cq values Cq satin Quantus Intensity RFU 5 10 15 20 25 30 35 40 45 Color by Sample x Dye SYBR Green vj Show all
95. UONBUWES FEM TT BORE 3 3 Automatic Quantification method e Analysis 30 x 70 EIE 3 amp Z Automatic Quantification method e Select Analysis Select Analysis Type Automatic Quantification Se ec7 Select Analysis Type x Automatic Quantification y Absolute and relative quantification using automatic CG calling a Fit Point Quantification Absolute and relative quantification using fit point CQ calling Tm Calling Melt temperature analysis Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping ry Spectra View Display of full spectrum optical data Cancel Absolute Quantification Settings Amplification Results as Table Samples
96. WPICgXIBUES 174 700 i MWP 3 000 x g 30 LightCycler Nano Instrument F Version 1 0 High Hesolution Melting EROFOIFS ZERIT 4 2 Q EERO op FTE IF ORAM COVE ISN L OL EIROFUT 92D PLAST OUGE EZ BEC SY 4 2 4 experiment 18 ERDER EZZEL S Run Settings Optics Settings ResoLight Intercalating yes TA x 700 4 4 v Of fe EIU SE Normal Quality gt Optimize Melt Acguisitions v Please selectthe brightest dye present in any well If vou are unsure use the Intercalating Dyes option Intercalating Dyes SYBR Green ResoLight i Hydrolysis Probes FAM VIC HEX Yellow 555 Red 610 TexasRed Cy5 Other Dyes The instrument can increase signal quality by acquiring for longer on each cycle of amplification after the hold is
97. amples asPlate Cq Values as Plate Note Excl Sample FAM Type Cq Quantity A 1000000000 7281 A 1 193 624 554 022 A2 2 5 1000000000 724 A 1 227 8567 724 473 A3 3 S 1000000000 7 348 A 1 163 534 446 445 Ad 4 S 100000000 10 726 A 107 788 708 927 AS 5 S 100000000 10 778 A 103 988 487 129 AB 6 S 100000000 10 787 A 103 325 148 965 AT S 10000000 14 173 A 9 723 883 046 A8 8 E 10000000 14 192 A 9 598 038 575 m 9 V 9 906 741 675 Y Bi Export gt gt gt EZI A PCHOSTAZIiwZLLESEHIBbICTSIfuEE EM TT i ORE Settings Quantifiers Amplification Selected Target Targetti Excluded early cycles 3 EJ Min Relative Amp 0 1 EJ Min Amp Quality 5 EJ Q Quantifiers E 2 1 2 Selected Target Target v Cq 0 2 5 5 T5 Loqio q Cq 3 3 logie q 37 24 r 0 9994 Error 0 225 E 2 009 Int Exclude Standards B Import Export Amplification
98. arget IS 1000 25 331 A 1 034 884 AS 5 Target S 10000 21 904 A 10 870 556 Ab B Unkn A Targetl u 26 432 Af 486 243 A 7 Unkn B Target u 33 845 A 3 002 AS 8 B1 g NTC Target e B2 10 BtdDNA 1E1 Target 10 31 789 A 12 304 B3 11 si EEE A is 100 29 054 A 80 418 Ra 12 dde 1nnn 25 AAR s qsn 14 7 BA Exon LightCycler Nano Instrument F Version 1 0 B 7 FKDEE EBI RC Tm Calling EA AXES BV E S Automatic Quantification Cq Values as Plate Cg Values as Plate 7 L Ghi 4Cqf 73 ESN CoD Eb VV TERARLET sp g Settings ZR IE ON 8E 3 d Vz X 2 4Zz1EU d se Automatic Range Results as Table Samples as Plate Cq Values as Plate 1 2 3 4 5 6 7 8 Display Settings A Q O O O Selected Dye SYBR Green Range Minimum 0 S Q C O O C Range Maximum 45 V Use Automatic Range uM E Q 9 O Each well is colored by its Cq for the selected dye The same Cq range and colors are used for amplification 4100000000 ae ES Cq within range C Empty Negative BS Cq too early Hl Cg too late se Automatic Range TV ar NF x 7 U Range M
99. complete Please select desired signal quality if you are unsure use the Normal Quality option High Quality Normal Quality a High Speed The instrument can automatically find the best settings to use during melting Ifthis option is selected the acquisition time will depend on dye intensity Ifthe option is not selected the same settings selected above will be used throughout the run V Optimize Melt Acquisitions ELM ERS TUT A EJ High Hesolution Melting EBOVOIFS ZERIT 4 2 2 Program Temp C Ramp C S Hold s Profile eG Programs 20 Temperature e Temperature Profile x 754 g g a e JL 25 0 0 08 20 16 40 25 00 33 20 41 40 50 00 58 20 01 06 40 01 15 00 Time mi
100. d 530 548nm 3 550 568nm StdDNA 1E1 4 570 588nm 5 590 608nm 6 610 628nm 7 630 648nm 8 650 668nm 9 670 588nm 10 690 708nm 11 710 728nm 4 00 nan B2 10 StdDNA 1E1 ES 44 UAR ARA S X SYBR Green Se ec eg Channels 530 548 nm Wells as 57 Wells as Plate gt gt Correct for background gt 00O LightCycler Nano Instrument F Version 1 0 Bl 7 KDEE EHI RC Tm Calling EA AXES BAEZ Automatic Quantification 2 3 Automatic Quantification 2 3 1 Automatic Quantification Fit Point Fit point Quantification 2 48 Fit Point iz Fit point Qua
101. duce noise decrease to improve resolution O by sample by genotype Color by Sample v gt gt gt LightCycler Nano Instrument F Version 1 0 High Hesolution Melting BEMI i ORE E Normalization Settings 2 amp Normalized 4 2 Bs amp amp d o gt Use Bilinear Normalizationd Tv a gt HBR BENOIT o 8 36588 ROB RA aE TOET gt Normalized 27 V 35V C Initial 7T OE SE C Final RERA Az 4 fb 5 C IEEE RACE Q IEXSILOEERSBII PEE HAE SEED LI FHI COOBEBEIZBERE SU BB CILE E T Raw Settings Normalization Settings V Use Bilinear Normalization Temperature Ranges Name Temperature 1 Temperature 2 Initial 68 765 71 582 Color by Sample v Temp Shift Settings 27 t Temp Shifted gt Apply Temperature Shift gt Temp Shifted 27M Intensity 7 es7o 9 Raw Settings Normalization Settings BLOSS Ra
102. eduction Range C 0 1 Ej Raw Normalized Temp Shifted iris Increase range to reduce noise decrease to improve resolution 73 74 75 76 77 78 Temperature Color by Sample pE I Samples as Plate Genotypes as Plate Pos Note Excl Sample Targett Baseline Genotype Color Name Note Al 1 325 E E uj A S Groupi A3 M c uy a E run Ad j Ac m A5 a AG Sample3 AT AB m M B2 10 Samplei ui roupi Export gt gt gt LightCycler Nano Instrument F Version 1 0 High Hesolution Melting da ORE results BEUF BICEDT Results as Table Samples as Plate Genotypes as Plate Difference Results as Table Differences Results as 7gp e 3 Raw Normalized Temp Shifted iis RFU Temperature C Color by sample D Getic gra Samples as Plate Genotypes as Plate Target Baseline Genotype Group2 Group2
103. er Nano Instrument F Version 1 0 Teb TANA TT ET EM TT i DEE 5 3 e Analysis 30 70 E AE Bz Automatic Quantification method OEE DIB S R8 EA s e Select Analysis Select Analysis Type gt Endpoint Genotyoing Se lect FA select Analysis Type j Wy Automatic Quantification Absolute and relative quantification using automatic CQ calling Fit Point Quantification Absolute and relative quantification using fit point CQ calling Tm Calling Melt temperature analysis A Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping ry Spectra View Display of full spectrum optical data Cancel Analysis Targets Endpoint Graph 7gge
104. inimum 21 Range 78x777777 32 Cq Cg too early Co too late Results as Table Samples as Plate Cq Values as Plate 1 2 3 4 H 6 7 8 l l Range Minimum 21 EE OES SSS mmewemm j O Use Automatic Range C Each well is colored by its Cq for the selected dye The same Cq range and colors are used for amplification 400000000 ze ES Cq within range C Empty Negative S Cq too early Hl Cg too late Display Settings Selected Dye SYBR Green v ERS TUT A pam lq F ADEE HERE Tm Calling EA AXES Fit Point Fit point Quantification 2 4 Fit Point Fit point Quantification 2 4 1 43 EE zt Automatic Quantification Fit Point Fit point Quantification 3 oFit Point Fit point Quantification method T4 5 fi OO d E24 EC R3 FIt Point 1og transformed curve Fit Point Fit point Quantificat
105. ion method a Analysis 30 SHEE Automatic Quantification method e Select Analysis 74 VR ACT gt EHR Select Analysis Type gt Fit Point Quantification Se ec7 Select Analysis Type f Wy Automatic Quantification Absolute and relative quantification using automatic CQ calling r7 Fit Point Quantification Absolute and relative quantification using fit point Ca calling Tm Calling Melt temperature analysis A Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping Spectra View Display of full spectrum optical data i Cancel Absolute Quantification Settings Amplifcation Results as Table Samples Analysis Stages Absolute Quantification Quantification Relative Quantification Quantifiers Amplification Notes Selected Target Target z Cycle Settings First Background Cycle 2 P D x
106. ity Chart Ratio heart kidney liver sorting order Group by samples v Chart Type Auto ooo LightCycler Nano Instrument F Version 1 0 X 4 4hAEIXCUZERLOILAPEZCHLTBeUZL s tSUNAMES ha OREN 3 4 4 ga ys7s Experiment 3 Run Settings Ui Profile Data Analysis Stages Relative Quantification Relative Quantity Chart 4 Analysis Select Analysis Wy Auto Quant Complete Sorting order Group by samples v Chart Type Auto M Targets Analysis Results Color Name Note Calibrator kidney La m liver MZ a H20 A _ Graph selected samples only gt Absolute Quantification gt Results74 F7IU7 Results as Table ze Cfi EER STCHEET To gt Excl LS Samples asPlate Cq Values as Plate Nate Excl Sample FAM Quantity 4 kidney AS 5 Ab B AT 7 A8 8 22 414 135 e gt Relative Quantification
107. ld s Prome e Programs 20 e Temperature Profile Temperature Profile 2 g a H E 2 Time minutes seconds Name 2 Step Amplification Hold 95 C for 600s No of Cycles 45 s Temp C Ramp Cis Hold s Acquire 2 Step Amplification 5 E E 35 5 i d Add Delete Mup Down add Dete up oom 88 LightCycler Nano Instrument Version 1 0 EROFAIAS gt PERI 5 2 3 Sg77D es Samples 23
108. lected the first one is displayed 5 eo000000 T I SSeS ES experiment bar Save Save gt Save 4s Save 45 27 C EZI A w X 4 hABEIXCUZERLOILAPEZCULTBerUZL s tSUNBWES FROP AIFS TERI 3 2 44 amp LightCycler Nano Instrtument 28 e Sg 7 Run E show log E Abort Run D BatRun _ Data 29 Data 3720277743 0
109. ler Nano Instrument F Version 1 0 PCROZIPF SY PLE LES ING EIEE KEW 77 iB OBIE Automatic Quantification Se lect m Select Analysis Type ES ag Automatic Quantification 2d Absolute and relative quantification using automatic Ca calling Fit Point Quantification Absolute and relative quantification using fit point Ca calling UN Tm Calling Melt temperature analysis Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping AJ Spectra View Display of full spectrum optical data Absolute Qug77 cg707 Settings Amplification Results as Table Samples XrL VER EELER EEZ SBUEUST S I Select Analysis Jor FID d SAM Nat BO fr TRIAD FERANES o MRE BII ZO Complete ERPRANSE C5FF Analysis Stages Absolute Quantification Relative Quantification rugs Quantifiers Amplification Selected Target Target v Excluded early cycles Intensity RFU Min Amp Quality 5 10 15 20 25 30 35 40 45 Cycle Color by Sample v Dye FAM v m Show all dyes CE S
110. nge C 1 5E Increase range to reduce noise decrease to improve resolution Results as Table Trl SamplesasPlate Tmas Plate Pos Note Excl Sample Target2 C1 J U B C2 18 5 StdDNA 1E1 IS 10 C3 19 S 100 C4 20 S 1000 C5 21 AE S 10000 CB 22 UnknA u Er 23 UnknB u C8 24 i D1 25 i emn gt gt gt EZIO AERA Bl 7 KDEE EHI RA Tm Calling EA AXES 75 Calling Settings Use Negative Derivation Noise Reduction Range 1 m AXIE EN ees Target Target2 v c V Use Negative Derivative Noise Reduction Range C 1 EJ Increase range to reduce noise decrease to improve resolution Q Peak Areas peak area peak area threshold P Peak Areas gt Melt Pegks Settings Peak Areas Melt Curve Notes Target Target2 yx
111. nstrument PCR b 1 2 1 T experiment bar D New LightCycler Nano Software New Experiment Open Experiments Unsaved Mew Experiment 2011 05 31 12 B New E Open 55 Save e Experiment Summary Name gt lt Enter gt Aofes Open Experiments Unsaved Dynamic Range HP uu p Mew IS Open Eal Save 35 Save As E Experiment 3 Run Settings Ui Profile Data Samples Analysis Experiment Summary Name Dynamic Range HP lt Instrument Type LightCycler Nano Instrument Id Experiment Created 2011 05 31 12 03 Run State Not Started Run Start Time Run Completion Time Settings Intercalating Dye Normal Quality Profile Analyses No analyses
112. ntification SYBR Green 1 Tm Calling 53 Tm Calling Analysis 30 AEEA Automatic Quantification 776 o e Select Analysis Select Analysis Type gt Automatic Quantification Select Select Analysis Type xi Wy Automatic Quantification Absolute and relative quantification using automatic CQ calling Fit Point Quantification Absolute and relative quantification using fit point CQ calling Tm Calling Melt temperature analysis A Endpoint Genotyping Genotyping using two colour endpoint fluorescence High Resolution Melt High resolution melt analysis and genotyping A Spectra View Display of full spectrum optical data Cancel Absolute Quantification Settings Amplification
113. ntification Settings Quantifiers Selected Target Target v Intensity RF U 2 5 5 Loqiofq Cd 3 3 logie q 37 24 17 0 9994 Error 0 225 E 2 009 Int Exclude Standards B Import Export Results as Table Samples as Plate Cq Values as Plate 530 Type Ca Quantity 5 1000000000 7 23 a 1 228 910 689 848 5 1000000000 7 236 A 1 224 110 885 678 5 1000000000 v 1 172 273 508 519 Note Excl Sample A2 5 S 100000000 v 103 740 399 3 AB 5 S 100000000 10 789 A 102 647 910 597 AT D S 10000000 14183 A 9 622 081 407 A8 8 S 10000000 1418 A 9 642 823 055 x nnn ro Y Q C al Results as 792 6 e results Results as Table K Cf amp f5428 9 7 Samples as Plate Cq Values as Plate 7L h CF SCONE 2 2 amp Bi s d E ERS T UT var PCROZIPF SY PLE LESION SEXIER ha OMEN Results as Table LightCycler Nano System 1x10 1x10
114. nutes seconds Mame High Resolution Melting Hold 95 C for 600s Initial Stage Temp C 65 Ej Se Amplification 45 cycles of 95 C to 60 C to 72 C Ramp C s 4 EJ Hold s 1 Led Hold 94 C for 60s Final Stage Temp C 95 EJ Hold 40 C for 60s Ramp Cts 0 05 EJ Hold s 1 Led a High Resolution Melting 65 C to 95 C at 0 05 C s ap Add Delete Up Dom LightCycler Nano Instrument F Version 1 0 High Hesolution Melting EROFOAIFS ZERIT 4 2 3 Samples Samples 4 23 Z0 777r 0 fg SE El gt Sample1 Sample2 Sample3 gt NTC Color Name Note HI Sample2 E Sample3 Te E NTC PUT WEDIIVICE WY CE 23x Z0 9 7 LO BSECEIUM CECB ESL Sg77O e7 gt Wells as Table Samples as ge A1 A4 A7 B2 B5 C3 C6 D1 D4 D7
115. on Fold Global Noise 4 Ej Pos JNeg Fold Global Noise 25 Ej LS PESA Samples as Plate CqValues as Plate Pos Note Excl Sample SYBR Green Type Cq Quantity C1 17 r Target2 N Cycle Settings Intensity RFU Thresholds C2 18 StdDNA 1E1 Target2 IS 10 34285 A 7 841 C3 19 Target2 IS 100 29 384 A 138 74 C4 20 Target2 S 1000 25582 A 1 289 012 Target2 V 8 710 904 Target Target D2 26 StdDNA1E1 Target2 iSi 10 34 355 A 7 522 Q KO Cirl a amp f amp C Results as Table ze C Fl Sf amp 2 200 d SN COVE TNEERLEF e results Results as Table ze CH SHR 47 Samples as Plate Cg Values as Plate ZLL h CH SColE 242 amp Bge 3 o B ERS T UT var lq F A OEE HERE Tm Calling EA AXES Fit Point E Fit point Quantification Results as Table Results as Table CHAR ITI AVY KFERAIOVY TVEC AEZ RARLETF KADY W Cq Caq Unkn A TILA Unkn Res
116. r of Background Cycles gt Number of Fit Po7 7 Quantifiers Amplification Selected Target Target v First Background Cycle 2 z E Number of Background Cycles 4 Ej Number of Fit Points 2 Ej V Automatic Quantification xFold Global Noise 4 PosJNeg Fold Global Noise 25 EJ Cycle Settings Thresholds gt gt gt EZI A EX Bl 7 KDEE EBHI RC Tm Calling EA AXES Fit Point Fit point Quantification Thresholds gt 4 o772 c 4 o772 74c Sree Quantifiers Amplification Selected Target Cycle Settings First Background Cycle 2 Ej Number of Background Cycles 4 s Number of Fit Points 2 Ej Thresholds VJ Automatic lt Quantification Fold Global Noise 4 EJ Pos Neg Fold Global Noise 25 ls gt REFE TIRET eld Automatic gt Amplification gt
117. rget2 Peak 1 StdDNA 1E1 10 100 1000 10000 StdDNA 1E1 Jile Bi Expor E Melt Peaks Melt Cyve results Peak Areas Melt Peaks Notes Target Target2 V Use Negative Derivative Noise Reduction Range C 1 n Increase range to reduce noise decrease to improve resolution 40E4 30E4 20E4 Temperature C Results as Table ALTE Samples as Plate Tmas Plate Pos Note Excl Sample Target2 Peak 1 C2 18 1i StdDNA 1E1 Unkn B UI 3 BtdDNA 1E1 1o 72 68 Q 4 KO Ctrl al 1 gt C Results as 79 6 Us esu s as Table CHE SHR 27 Samples as Plale Tm as Plate 7L h C f 8Tmfe 7 Bg 4 lq F A OEE HERE Tm Calling EA AXES Tm Calling Results as Table Results as Table GE C fd Aa ZR 7 VS 0C 7 AML x VW AE SNE 7 O Bi i Tm
118. ults as Table Samples as Plate Cq Values as Plate D2 D3 Note Excl Sample SYBR Green EM Target2 StdDNA 1E1 Target2 Target2 Target2 Target2 Target2 Target2 NTC Target 26 StdDNA 1E1 Target2 27 SUDNATE2 Target Quantity Qo 34 355 7 522 29 276 A 147 827 Y Cq Values as Plate amp Export Co Values as Plate CZL HF CH 2Cgfi Cq so gy Settings Use Automatic Range Lo Results as Table Samples as Plate Cq Values as Plate 00000000 00060000 Display Settings Selected Dye SYBR Green M Range Minimum D n Range Maximum 45 n VJ Use Automatic Range lt p Each well is colored by its Cq for the selected dye The same Cq range and colors are used for amplification graphs ES Co within range E Empty Negative S Cq too early Hl Cg too late LightCycler Nano Instrument Version 1 0 lq F A OPE HERE Tm Calling EA AXES Tm Calling 2 5 Tm Calling 2 5 4
119. w Normalized ELTE Difference Notes _ Apply Temperature Shift e Intensity Threshold 005 Iz ni 5 gt 65 70 75 B0 85 90 Temperature C Color by Sample v gt gt gt EZI A EU High Hesolution Melting BEM 77 iO BE e7erce Q EHR e eT Uv TU CA AMEBEE GU TT TOR EBREBRAECET 0 Raw Normalized Temp Shifted Eis Notes 0 4 0 3 0 2 LL c 0 1 0 0 1 j 73 74 75 76 77 78 Temperature C Color by Sample Z Results as Table gagse 77e Results as Table Samples as Plate Genotypes as Plate Pos Note Excl Sample Target Baseline Genotype AG 6 Sample3 uj C AT 7 a u v B1 g i Sample3 uj B2 10 Samplet uj B4 12 Sample3 uj B5 13 D ui A ATTMEIUCU MES DLN HIB N X71T EU CfEbh Notes 7I IO ERE RRS Q 9x High Res Melt No baseline well selected using well no 1 instead LightCycler Nano Instrument
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