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ウシ白血病ウイルス検出用Probe/Primer/Positive control

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1. http wwwr takara bio coJp 16 CY415 VII Cycleave PCR Reaction Mix SP CY510 NucleoSpin Blood QuickPure 740569 10 50 250 Thermal Cycler Dice Real Time System TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 96well Hi Plate for Real Time NJ400 Sealing Film for Real Time NJ500 Plate Sealing Pads 9090 48 well snap plate NJ700 Flat cap for snap plate NJ720 0 2 ml Hi 8 Tube NJ300 0 2 ml Hi 8 Flat Cap NJ302 0 2 ml 8 strip tube individual Flat Caps NJ600
2. Cycleave TaKaRa Ex 7gg Thermal Cycler Dice http wwwr takara bio coJp 17 CY415 Iaf4R4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJjp v201407Da
3. 4 PCR a PCR Auto PCR Manual V V 1 2 DNA DNA DNA NucleoSpin Blood QuickPure NucleoSpin Blood QuickPure NucleoSpin Blood QuickPure 1 200 ul Proteinase K 25 ul 1 5 ml Buffer BQ1 200 10 20 70 15 2 96 100 200 3 NucleoSpin Blood Column Collection Tube 2
4. Ct CP Positive Control Ct bs C CH CFP Positive 0 Control CHECSDM CH SDM LEH 5DSDM CP YSDM SD SDSEiM CP SDM CP SDM NIC _ FAM std 10 std 107 std 103 std 104 std 10 std 108 Sample 1 M M M Sample 2 Sample 3 FAM Sample4 FAM 2997 2992 Sample5 FAM z2oxl221xio zisxel2xio 3 izoxel ioxo 1 FA FA FA FA FA FA FA FA FA FA FA FA
5. CycleavePCR Reaction Mix SP CY510 PCR Hot Start PCR 7 3 g Ex Tag Hot Start Version RNA DNA RNase H RNase H RNA
6. tO complete a ran The FER reactinhs contail CR Check http www takara bio co jp 11 CY415 3 Setup Plate Setup Define Targets and Samples Target name Reporter FAM Quencher None Assign Targets and Samples Passive Reference ROX 7500 Fast Real Time PCR System StepOnePlus He kr EF mrmt me the mmm tt Famiman Mari EMparmment Crz4HE 120El TF UF NHme Targots nid Spample3 Aseior Fargets ard am ED en ne Cm BF ni li Th hk Fd Fr sir biti snarlinnmi i Dr Ta EhTmweefTt TE tid CE Owne Ta ed Sarmgiat Al Targels ad mlgs Te FE ff iE LE mm EE im ei am TTF HFP TFT a TE HH mL tIWhulFPmEMNIELEIEWE IE nH Wi We Ta en nm mei ie Carmrm FEF RF EE nas gt 1 http www takara bio coJp 12 CY415 4 Setub Run Method Stage 1 Hold Reps 1 95 10
7. 1 1 RNase H OE NA 1 25 ul X 50 1 Probe Primer Mix for BLV 5 xX 1 250 ul 2 Positive Control 2 Xx 10 copies ul 2 100 ul 3 EASY Dilution for Real Time PCR 1 ml 1 2 1 2 3 http wwwr takara bio coJp 2 CY415 Probe Primer Mix for BLV
8. 2 Positive Contrtol Thermal Cycler Dice Real Time System 1 2 x CycleavePCR Reaction Mix SP 12 5 ul 1 x Probe Primer Mix for BLV 5 Xx 5 ul 1 x or Positive Control or 5 ul 2 RNase Free dH20 2 5 ul Total 25 ul Life Technologies PCR 1 2 x CycleavePCR Reaction Mix SP 12 5 ul 1 x Probe Primer Mix for BLV 5 Xx 5 ul 1 x or Positive Control or 5 ul 2 ROX Reference Dye or ROX Reference Dye ll 3 0 5 ul RNase Free dH20 2 ul Total 25 ul 1 CycleavePCR Reaction Mix SP CY510 2 100 ng 3 7300 RealTime PCR System StepOne StepOnePlus RealTime PCR System ROX Reference Dye 7500 Fast RealTime PCR System ROX Reference Dye ll 1 0 5ul 2 5 ul 1 1
9. STD Omit Omit 5 zax Positive Control Positive Control a EE http www takara bio co jp 8 CY415 5 lt gt sr PIPES a t SD Gt CP Gt SDM a Ct SDM
10. 11 000 xX 9 1 4 NucleoSpin Blood Column Collection Tube 2 m Buffer BQ2 2 350 ul 11 000 X g 3 5 1 5 ml 70 Buffer BE 50 ul 1 11 000 X 1 1 Proteinase K 740569 10 Proteinase K 6 mg Proteinase Buffer 260 ul 2 Buffer BQ2 Wash Buffer BQ2 concentrate 3 ml 12 ml 96 100 http wwwr takara bio coJp 4 CY415 V 2 1 1 o 20 ul
11. 4 http wwwr takara bio coJp 3 CY415 IV 1 PCR 2 SE 3 3 1 2 3
12. Thermal Cycler Dice Real Time System 2 PCR FAM FAM Hold Cycle 1 95 10 2 step PCR Cycle 40 95 5 64 30 Hgld Tn rir LE ata Coalition 3 ta Hun http wwwr takara bio coJp 7 CY415 4 UNKN STD NTC Dye 2 n 2 n 2 n 1
13. http wwwr takara bio coJp 9 CY415 7500 Fast Real Time PCR System Life Technologies 7500 Software for 7500 Fast Rea Time PCR System StepOnePlus Real Time PCR System Life Technologies StepOne Software for StepOne and StepOnePlus Rea Time PCR System 1 New Experiment Home Advanced Set up 7500 Software li Advanced Set up Fram Temetate http www takara bio co jp 10 CY415 2 Setup Experiment Properties Experiment name 7500 Fast Rea Time PCR System 7500 Fast 96 Well StepOnePlus StepOnePlus Instrument 96 wells Quantitation Standard Other Standard 2 hours to complete a run 7500 Software Otherl gt Otherl EH
14. TP900 TP960 Thermal Cycler Dice Real Time System Lite TP700 TP760 Thermal Cycler Dice Real Time System Software Applied Biosystems 7500 Fast Real Time PCR System StepOnePlus Real Time PCR System Life Technologies 0 2 ml PCR 0 2 ml 8 strip tube individual Flat caps NJ600 1 8 0 2 ml Thermal Cycler Dice Real Time System CycleavePCR Reaction Mix SP CY510 1 2 Xx CycleavePCR Reaction Mix SP 625 2 RNase Free dH20 500 wl 3 ROX Reference Dye 25 ul 4 ROX Reference Dyell 25 ul DNA DNA NucleoSpin Blood QuickPure 740569 10 50 250 1 5 ml 1 000 200 20 10
15. CY41 IafHa Probe Primer Positive control lt CY224 gt Cycleave PCR BLV CY224 CycleavePCR Reaction Mix SP CY510 CY415 PCR V 4 PCR v201407Da bovine leukemia virus BLV B DNA BLV tax PCR Probe Primer Positive control PCR DNA polymerase Tli RNase H
16. Ef mm He er CF ler dH Stage 2 2step PCR Reps 40 95 5 64 30 Sample Volume 25 ul 5 Start run 6 Analysis Amplification Plot Analyze tax Threshold Baseline Auto 14 Auto Baseline Amplification Plot Option Auto Option Threshold Auto Auto Baseline Threshold Auto Auto Baseline Analyze Baseline http wwwr takara bio c
17. Positive Control Positive Control PSFLEmEEI be ur ke 1 1 am her EES hs Dn LT wigw Flate Layaut Wiew Well Table Select Wells Wh Balactltem w alart lem Show In Tahle WF GTnUH By Wa alTlhla a Tardet ame Task gs CT hean uahtity atlty 7 BL TE indeterrmi BL TMANMDARD B B29 H7 1H 5 BL TANMDARD 33 3 54 1II BL Ee a 2 1 HI F5 BL TANEARD 3 313 3 5 31 1 BL Ta AR 23 121 23 21 1II IIh HS BL TANMEaRD 1 5HH 19d 59 1 HHHHI 5 aITIhla 1 BL UNRIIGWHI Unrdaterrml CE alTihlg 2 BL HEREIGWWI 5 1 g HH1 B 7 BB alTihle 3 BL URIGMWWI 1 551 1 551 B77 72 JH 7 lt gt Analysis Amplification View Well Table t Ct Positive Control Ct Ct Positive Contrtol
18. 2 Positive Control 2 Xx 10 copies ul 3 EASY Dilution for Real Time PCR Positive Control V 2 Positive Control 1 5 ul 1 Positive Control 2 X 10 copies ul EASY Dilution for Real Time PCR 2 1 5ml 5 2X104copies ul 2 X 103 copies ul 2 Xx 10 lt copies u 2 X 10 copies 2 Xx 109 copies ul 3 1 5 ml EASY Dilution for Real Time PCR 90 ul 4 Positive Control 2 X 10 copies uu 10 ul 2 Xx 10 copies 5 2 X 104copies ul 10 ul 2 Xx 103 copies ul 6 20
19. http wwwr takara bio coJp 6 CY415 V 4 PCR 3 PCR Thermal Cycler Dice Real Time System 7500 Fast ReaL Time PCR System StepOnePlus Real Time PCR System Life Technologies V Thermal Cycler Dice Real Time System 1 OK EEEH lt gt Thermal Cycler Dice Real Time System Software lt Absolute Quantification gt
20. 3 3 Positive Control 3 Positive Control 1 4 PCR lt gt DNA 1 DNA 100 ng http wwwr takara bio coJp 5 CY415 V 3 Positive Control
21. BLV tax tax Positive Control tax A2eo EASY Dilution for Real Time PCR DNA DNA TE tRNA rRNA 20 PCR Thermal Cycler Dice Real Time System
22. oJp 13 CY415 opfions UtO Auto Baseline Target BLV Threshold to 0 333035 7 Auto Baseline 5howr Threshold Baseline 5tart Well Tarnet Baseline End Well Tar Bt 4 4 I optioms Auto Auto Baseline Target BLV Threshold Aulo 0 333035 EE Jute Baseine Show Threshold Baseline Start Well Target A Baseline End Well Tarnet 8 Auto Baseline Analyze Target BLV Threshold Auto 0 333035 to Baseline Show Threshold Baseline Start Well Target 4 Baseline End Well Target 4 ee BL Auto af ET EE TE Ta En mt TE Lr WCSRm rh Hm ri TE LF HOmHEE rs Fa WF LTE EE rn RE Fe em rm 1 et Tn a Te ra EET HIHail aa mm 1 TmrE m PPmmwf Ti mm mts Ta LS TPmmmhed ap EE PT FT mmm 5 nL Er Ah T3 1 http ww
23. w takara bio co jp 14 CY415 Baseline well Analysis Setting Advanced Setting Baseline Setting for well well Baseline Setting to use Use Ct Setting for Target well Baseline Start Cycle End Cycle HH ot Ee 5 HI mWTHITEHE Fn UHFG We FRTH ST ERrTHHE la hor HH THB HHmdI JEmm nm PTP Tor ee TL mh a a mim LT SigCt We and Tarnewi Eliie Str Tnf i LN Tarat E Baswls am Ets linn End Rai Sa i Use ED Ue CT ati Dafitred tor Ta PErTL HRPHTEH Edi SEC Ed bc fF Baseline SetUp Plate SetUp Define Targets Quencher TAMRA http www takara bio co jp 15 CY415

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