Cycleave®PCR O-157 (VT gene) Screening Kit Ver.2.0
Contents
1. 3 4 3 1 2 3 5 PCR2. PCR VT2 VT2 VT2 http www takara bio coJp 15 CY217A VII VII 1 Takao T Tanabe Y M Hong Y Shimonishi H Kurazono T Yutsudo C Sasakawa M Yoshikawa and Y Takeda 1988 Identity of molecular structure of Shiga like toxin VT1 from Esherichia coli O157 H7 with that of Shiga toxin Microb Pathog 5 357 369 2 Jackson M P R J Neil A D O Brien R K
3. 1 OUT Q gt 2 DNA OUT Q PCR ROX F FAM Primer Probe Mix DNA 3
4. FAM ROX IC d 2 ta pu m E E e i n e 3 4 B 3532 M 18 18 20 22 M 74 29 Mp TP 14 C 1 40 dO Ld JA dA 33 pul 8 amp dH 0204 H4 7 O00 208 24 24 20 12 d 34 ON AD 42 44 4 GU 5 4088 PELET 3 gt http www takara bio coJp 9 CY217A 6 gt lt lt N gt P gt OK OUT gt Posi Nega ND PCR Error
5. 2 5 1 2 FAM IO ROX BREl Auto http www takara bio coJp 8 CY217A 2 NC PC NC N gt FAM FAM ROX IC d fj H e m E Pir T a m s z 5 4 B 5324 18 18 20 22 4 24 29 6 10 4E I D uai dA dh ENT E ROHOIM T NM CAE I3 1d M CONG RE IEEE SEET CREUT PC P gt FAM ROX
6. 1 VT1 2 VT2 VT2vha VT2vhb VT2vp1 171 bp 1 2 3 4 8 2 X Cycleave Reaction Mixture 4 PCR DN PCR s PCR zw EXT VT1 VT2
7. 3 VT1 VT2 RE 1 Q 2 X Cycleave Reaction Mixture 12 5 ul 1X VT Primer Probe Mix 5 X conc 5 ul 1x or or WEK 5 up Q dH20 2 5 ul Total 25 ul DNA 0 3 2 3 PCR 1 http www takara bio coJp 6
8. http www takara bio coJp 3 CY217A Il 20 C 1 3 4 8 20 I PCR Thermal Cycler Dice Real Time System III with PC TP970 Thermal Cycler Dice Real Time System 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 Life Technologies 95 200 ul 20 ul 10ul IV 1 PCR 2
9. Hold Cycle 1 95 C 10 3 step PCR Cycle 45 95 C 5 55 C 10 72 25 Dies 1 8 Start http www takara bio coJp 11 CY217A 9 Analysis Amplification Plot Threshold Baseline Manual 1 Amplification Plot Options Target VT Target Threshold Auto Auto Baseline Show Threshold Baseline E 1 b c 1 DD wget VT WE Theeshote mm 00001 Ruin asaina mw Trushaa F Bastina Exit ell Tegola Bacene End Von B Target a 2 Analysis Setting Analysis Settings http www takara bio coJp 12 CY217A 3 Analysis Setting Select a Target VT Target Ct Settings for Target Name Threshold Baseline
10. Apply Analysis Settings x Threshold Amplification Plot Log Baseline Amplification Plot ri Tries for ERD Duni rns ara b CT Suf ings Fog Sag Advanced Samus g Eiaa be Enalbop naa Ue arant S carpe rm Td dopdcrh ant TE x Fa ete bi mz i oe Dena rl npe m To usa e iga Dn a equ bb gn Top cape Roms Fe labis cecus axi Lee Ch Ebrga Es ihe bedaga tus sew capias Detail i Sem Dub rt irt aa To cg icu dr Tat CE Er ror nrbes FE agnos To enc Et dz ei eed ras mh EO CF Debet IT DawieecuECHa amp UTO ei TE pai AUTO EE DEHE Deu Abel ai Tue get Ls Sette Fon VT Tug Pacina zx aum fiT HI a Do5 esu jns r Uce Dat DR 8 33 c larg AJT a nm IER T TE Mi LB d Bie mac Duselee Ded Cue i ed itm 100006 Threshold x H a bar Li PLA E 4 Baseline d B DI Ti 5 00001 Tape VT Thnrshold Amn 3000 C Auto Baseline howr v Tawahola v Baseline Start Well Targat Barehna End Well B Targat http www takara bio coJp 13 CY217A
11. VT Primer Probe Mix FAM ROX dH20 ELK CS NaOH Solution 50 mM NaOH Tris HCl Buffer pH7 0 dH20 for Dilution VT1 Positive Control VT1 VT2 Positive Control VT2 Lot AK2701 Lot AK2601 v201412Da
12. PCR Auto PCR Manual http www takara bio coJp 4 CY217A V V 1 1 2 PCR 3 i RI POR h LR 4 BT 2 DNA 100 ul 1 5 ml 2 0 ml 106 000 X g 10
13. VIT VT2 PCR PCR DNA DNA DNA 3 1 100 PCR PCR Hot Start PCR prie Ex Taq HS A NN RNA DNA RNase H
14. CY213 VT1 VT2 026 0103 0111 0121 0145 0157 1120 1 EO 1 RNase H NA 1 http www takara bio coJp 2 CY217A l 50 25 Q 1 2 X Cycleave Reaction Mixture 2 X conc 625 ul 2 VT Primer Probe Mix FAM ROX 5 X conc 250 ul Q 3 dH20 1 ml NaOH Solution 50 mM 1 5 ml x 4 O 5 Tris HCl Buffer pH7 0 1M 500 ul x2 Q 6 dH20 for Dilution 1 ml 7 VT1 Positive Control 150 ul 30 8 VT2 Positive Control 150 ul 30
15. FAM VT1 VT1 4 VT1 VT2 VT1 5 3 3 VT2 ROX FAM VT2 VT2 4 VTI VT2 VT2 6 3 4 ROX TERE VT1 VT2 VT1 VT2 VT1 2 ET NS z b NAR s 4L T2 dz 223 Vas 3 x1 x2 3 4 VT1 VT1 VT1 5 6 x7 VT1 VT2
16. 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 1 ml 8 strip tube amp cap Set NJ903 0 1 ml 8 strip tube individual Flat Caps NJ902 0 2 ml 8 strip tube individual Flat Caps NJ600 0 1 ml 96 well plate for Real Time NJ900 http www takara bio coJp 16 CY217A Cycleave TaKaRa Ex Taq Thermal
17. 10 View Well Table Shimi i Cin Alutiz HL Ul Ras Oma B EFL MME ES Nunipie PENE Wiii a Jd M i PIE Na Target Name LI MEZ Applied Biosystems 7500 Real Time PCR System StepOnePlus Real Time PCR System Life Technologies StepOnePlus Real Time PCR System ROX target ROX IC FAM ROX target http www takara bio coJp 14 CY217A VI 1 ROX FAM 1 1 VT1 VT2 2 3 2 VT1 ROX
18. A CS filis PASTE SE Ens f Bt ORE XEBURUACUSE BEC EH S C DERE RNase H RNA 1 VT1 VT2 FAM ROX ERA scc a UR LOU RE S m CycleavePCR O 157 VT gene Screening Kit
19. 50 mM NaOH Solution 85 ul 100 10 lt 1 M Tris HCI pH7 0 O 15 ul lt 2 000 10 000 X g 10 fii d A VT1 VT2 1 5 ul http www takara bio coJp 5 CY217A V 2 1 VT1 VT2 VT1 VT2 1 1 a 20 uu 1 5 ul
20. Cycler Dice Real Time System 2 PCR 3 Sieg PCA Hold 3 Cycle 1 95 C 10 3 step PCR Pi Cycle 45 95 C 5 55 C 10 72 20 Aric 50 55 1n LE DISCE Ek pigs nni nn 3 Speed I Thermal Cycler Dice Real Time System III TP970 Normal C Thermal Cycler Dice Real Time System TP900 Lite TP700 Fast Speed http www takara bio coJp 7 CY217A 3 mm 4 Omit Cizigj amie 1 1 15 9 ROX
21. Holmes and J W Newland 1987 Nucleotide sequence analysis and comparison of the structural gene for Shiga like toxin Ouand Shiga like toxin Il encoded by bacteriophages from Escherichia coli 933 FEMS Microbio Lett 44 109 114 3 Ito H A Terai H Kurokawa Y Takeda and M Nishibuchi 1990 Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91 H21 isolated from a patient with the haemolytic uremic syndrome Microb Pathog 8 47 60 4 Weinstein D L M P Jackson J E Samuel R K Holmes and A D O Brien 1955 Cloning and Sequencing of a Shiga like toxin typell variant from a Escherichia coli strain responsible for edema disease of swine J Bacteriol 170 4223 4230 Cycleave PCR O 157 VT1 VT2 Typing Kit CY222 Cycleave PCR EHEC 0157 026 Typing Kit CY237 Cycleave PCR EHEC 0111 0121 Typing Kit CY238 Cycleave PCR EHEC 0103 0145 Typing Kit CY239 Cycleave PCR EHEC 0165 Typing Kit CY241 EHEC O antigens PCR Typing Kit RR133A Thermal Cycler Dice Real Time System III with PC TP970 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 Adhesive NJ501
22. CY217A V 3 PCR 3 PCR 3 vp e Z Z Tik Thermal Cycler Dice Real Time System a Applied Biosystems 7500 Fast Real Time PCR System Life Technologies Thermal Cycler Dice Real Time System 1 lt CycleavePCR Kit gt SHARUBIISE Be T a EET FIJE iC vele as PCR Ki i n nes SNPs CycleavePCR Kit Thermal Cycler Dice Real Time System Software PM M Plus Minus Assay gt gt Thermal
23. Cycler Dice Idf4R4 Tel 077 565 6999 Fax 077 565 6995 http www takara bio coJjp 23J2 UA ERU v201510Da
24. ND Q http www takara bio coJp 10 CY217A Applied Biosystems 7500 Fast Real Time PCR System Life Technologies 1 Advanced Setup New Experiment 2 Experiment Properties Quantification Standard Curve TaqMan Re agents Other Other Include Melt Curve 3 Plate Setup Define Target Target Name VT1VT2 Reporter FAM Quench er none 4 Plate Setup Define Target Target Name IC Reporter ROX Quencher none 5 Define Samples NC PC 6 3 4 5 Plate Layout Passive Reference none 7 Instrument
25. x 1 CY217A Tahaha Cycleave PCR O 157 VT gene Screening Kit Ver 2 0 026 0103 QO111 0121 0145 0157 1120 1 Lot AK2701 3 dH20 5 Tris HCI Buffer pH7 0 3 v201510Da 0157 H7 EHEC EHEC EHEC 0157 H7 EHEC
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