取扱説明書 - 東洋紡ライフサイエンス事業部
Contents
1. V1 PCR 4 slope lt 3 95 2 PCR PCR Tm Tm2. DNA 3 GC tail Tm
3. DNA DNA DNA DNA
4. PCR DNA DNA PCR DNA PCR DNA DNA
5. 2 Tm 2 Tm 4 10 SYBR Green PCR 1ml x 1 QPS 201T 40 SYBR Green PCR THUNDERBIRD SYBR qPCR Mix 1 67ml x 3 200 THUNDERBIRD SYBR qPCR Mix 50 x ROX reference dye 50ul 1000 QPS 201 x 5 QPS 201 5 QPS 201 PCR 1ml x 1 40 QPS 101T
6. 3 2 8 C 20 C 10 SYBR Green 50 x ROX reference dye Applied BOS VIS Agilent Technologies 4 1 20 C 2 8 C 20 C ROX reference dye
7. PCR 2 step PCR 98 C 10 60 C 10 68 C 30 500bp 40 cycles 98 C 10 68 C 10 68 C 30 500bp 40 cycles 2 step PCR 98 C 10 68 C 30 500bp 40 cycles 0 98 C 10 60 C 10 68 C 30 500bp 40 cycles 98 C 10 60 C 10 68 C 1 500bp 40 cycles
8. Tm 2 10 1 2cm 3 GC 10 KOD DNA polymerase GC 70 4 PCR
9. GC Tm Tm Tm Tm 2 Tm 5 C SYBR Green
10. 3 GC CDNA DNA HPLC OPC 1 Tm Nearest Neighbor method Tm 50mM Na 0 SuM Nearest Neighbor method Tm
11. http Wwww toyobo co jp bio s 3 3 DNA DNA cDNA genomic DNA plasmid DNA virus DNA DNA a cDNA cDNA PCR 10 PCR PCR ReverTra Ace qPCR RT Kit Code FSQ 101 ReverTra Ace qPCR RT Master Mix Code FSQ 201 ReverTra Ace qPCR RT Master Mix with gDNA Remover Code FSQ 301 PCR 20
12. ASP Allele specific primer PCR SNP Single nucleotide polymorphism ASP PCR GC ASP PCR 3 SNP GC tail GC tail Tm 100bp ASP PCR 4 3 2 SNP 3 3 A T G A 2 Tm 3 C 3 C GC tail
13. 16 3 3 1 5 3 3 1 3 3 0 75 60 65 70 75 80 85 90 95 860 65 70 75 80 85 90 95 60 65 70 75 80 85 90 95 Temperature C Temperature C Temperature C 100bp 341bp 3 2 Tm 100bp Tm 79 C 341bp Tm 84 C 3mm PCR 50mM NaOH 180nl 98 C_2 Vortex 95 C 10 1M Tris HCI pH8 0 20ul
14. 60 C 4 2 PCR PCR PCR Ct PCR Ct Ct PCR Ct PCR Ct Ct PCR 110
15. 141 8633 10 2 TEL 03 6422 4819 FAX 03 6422 4951 E mail order lifescience toyobo jp TEL 06 6348 3888 FAX 06 6348 3833 9 00 12 00 13 00 17 00 E mail tech_osaka toyobo jp URL http Www toyobo co jp bio
16. 1 50mM NaOH 180ul Vortex 95 C 10 3mm 1M Tris HCI pH8 0 20ul 20mg Vortex 20mg 5mg 12 000rpm 5 optional 20pul 0 5ul 2ul 2 3mm BufferA 100ul Buffer A Vortex 100mM Tris HCI pH9 5 95 C 10 1M KCI Vortex 10mM EDTA 1710 20ul 0 5ul 2ul Buffer A 4 1 50 Hl 2
17. SYBR Green PCR lt gt uM MM _ 0 2M x bp bp 02M
18. KOD Plus KOD FX KOD Dash PCR 2kb 2kb melting temperature Tm 60 70 C GC 45 60 Forward Reverse 3 3 GC 3 G C
19. Melting Dissociation Curve Analysis 1 98 C 2 2 50 C 68 C 3 500bp 30 30 45 lt 60 30 500bp 500bp 30
20. 50 x ROX reference dye KOD SYBR qPCR Mix KOD SYBR qPCR Mix ROX 41 1 ROX 1 x qPCR Mix 50 x ROX 1 67ml 66 8ul QKD 201 1ml 40ul QKD 201T ROX 0 1 x qPCR Mix 50 x ROX 1 67ml 6 7ul QKD 201 1ml 4ul QKD 201T 3 1 PCR PCR 2 SYBR Green
21. Tm GC tail GC tail P 16 SNP a lt C Primer 5 cc GC tail Tm w Primer GC tail 4 SNP Tm 3 C Primer Primer 3 ATA 5 GC tail Tm 5 3 4 SNP 1 2 1 Tm 5 C Tm 5 C 50bp
22. 1 3 4 Bio Rad MiniOpticon 2 0 Bio Rad MiniOpticon 1 Create a new run 2 Create New sl jnset St sarmple YolurmE Est Run Fime dB8H Cer FD Er ft ED tor Flate Regd 2 mam MTs hes 3 x ba mime 5 MaltCune 5 ta Cincrement hb5 CE fer 8 a Fe Insert Melt Curve END nd Add Plate Read tn p x 4 1 Sample Volume pl 2 2 step Insert Step 3 step 3 Insert Melt Curve GC 99 C 4 Add Plate Read to Step
23. 3 19 5 1 GC 1 KOD FX KOD Plus PCR TGF 1 9kb Human genomic_ DNA gDNA gDNA 10 5 no template control NTC PCR 98 C 2 98 C 10 60 C 10 68 C 120 40 cycles 30 500bp ABI StepOnePlus Taq DNA polymerase THUNDERBIRDY SYBR qPCR Mix 1 9kb 1kb DNA Ladder KOD FX Neo KOD FX KOD Plus Ver 2 KOD SYBR qPCR Mix THUNDERBIRD SYBR qPCR Mix
24. 4 Ct 50 cycles 5 SYBR Green DNA SYBRY Green PCR GC 80 99 C b PCR
25. do 200Ul 2 48Ll 20MI 5Ll 20LI 5hl 6 st 60 65 70 75 80 85 90 95 60 65 70 75 80 5 90 95 Temperature Temperature C 19 8 RNA cDNA cDNA PCR ReverTra Ace qPCR RT Kit Code FSQ 101 ReverTra Ace qPCR RT Master Mix Code FSQ 201 ReverTra Ace qPCR RT Master Mix with gDNA Remover Code FSQ 301 PCR cDNA ReverTra Ace qPCR RT Kit PCR cDNA
26. no template control 50 x ROX reference dye 50xROX reference dye SYBRY Green 4 1 50 x ROX reference dye PCR 45 60
27. 5 Fast Mode 50 x ROX reference dye Applied Biosystems ROX 2 QKD 201 QKD 201T KOD SYBR 5ml 1ml 2 8 C 3 qPCR Mix IO 2 1 67ml x 3 1mlx 1 50 20C 2 8 C ROX reference dye 25ULl SULl KOD SYBR qPCR Mix SYBRS Green dNTPs Mg KOD exo DNA polymerase DNA 2x DNA cDNA genomic DNA plasmid DNA 1x ROX passive reference dye 20 C
28. DNA SYBR Green DNA SYBR Green 1 DNA Ct PCR PCR cDNA PCR DNA DNA
29. yeast RNA PCR 110 slope gt 3 1 no template control no template control NTC 4 2 PCR PCR HPLC
30. 1 PCR 10 b genomic DNA virus DNA 9enomic DNA virus DNA 50ul 200ng 2 SYBR Green 2 DNA 2 DNA DNA SYBR Green genomic DNA c plasmid DNA plasmid DNA plasmid DNA DNA
31. i 1 Tm Tm 64 9 41X yG zC 16 4 wA xT yG ZG w x y Z A T G C Wallace RB et al 1979 Nucleic Acids Res 6 3543 3557 Sambrook J and Russell D W 2001 Molecular Cloning A Laboratory Manual Tm http www toyobo co jp bio 2 Tm Tm 2 Tm PCR 15
32. 98 C 10 50 C 10 68 C 30 500bp 40 cycles 1 ABI StepOnePlus VeriFlex Bio Rad 3 1 Applied Biosystems StepOnePlus 2 2 2 Applied Biosystems StepOnePlus 1 Design Wizard Advanced Setup QuickStart 2 reagents SYBR Green Reagents Design Wizard Methods amp Materials Advance Setup Setup Experiment Properties QuickStart Experiment Properties 3 Run Methods Graphical View abular View 1 Reactinn wolurme Per wyell 20 HL Add Stage AddBteh i Undn Set Termperature fl
33. TPgfatuFg PC TemEigfgtuFE PE 2 GC GC 70 PCR ReverTra Ace qPCR RT Kit Code FSQ 101 HeLa total RNA cDNA cDNA 5 5 PCR PCR 98 C 2 98 C 10 60 C 10 68 C 30 40 cycles ABI StepOnePlus THUNDERBIRD SYBRY qPCR Mix GC 709 8 IGF2R GC 83 cDNA A PCR KOD SYBR qPCR Mix GC
34. Applied Biosystems 7500 Fast Real Time System 1 Instrument 2 Thaimal Eurlgr Frntnrnl Thermmal Frghilg aug Increment Ramp Riate Stage 1 taga 2 ta ga 3 9 5 1 1 1 a so 50 0 ln sa lt Add Cicle Add Hald Settings 5amhle nlume fu el Nl Fuh hiode Fast rhHH Espet gde 5gleclywiEwr Fillal Dala Collection lslage 2 Step 3 658 0 E30 lt 1 Sample Volume uL 2 3 GC 99 C GC 80 3
35. Tm GC GC E 2 GC tail Tm 100bp Tm Tm GC GC tail Tm GC tail Tm Tm Tm Tm Tm
36. 3 3 Roche LightCycler Nano 1 0 Roche LightCycler Nano 1 New 2 Experiment Name 3 Run Settings Intercalating Dyes 4 Profile 1 Program add Hold 2 98 C 120 5 C 3 Program add 3 Step Amplification 4 5 Acquire WC Name 3 Step Amplification No of Cycles TaFTiB rc Ramp Ci Hald 1 CALIE Add Bmlete Up Down 6 Program add Melting 7 50 00 Time minutes seconds 5
37. 70bp 150bp Wild type WT Knock in KI Primer Primer wr 79bp 150bp Tm 70bp 150bp Tm P 15 Tm 3 C 5 C 500bp 300bp Tm 3 C Primer Primer 4 WT Dn Tm 3 C rE Fro KI 5 C Primer Primer
38. PCR THUNDERBIRD Probe qPCR Mix 1 67ml x 3 200 THUNDERBIRD Probe qPCR Mix 50 x ROX reference dye 50ul 1000 QPS 101 x 5 QPS 101 5 QPS 101 cDNA PCR cDNA ReverTra Ace qPCR RT Kit PCR cDNA mm reozor 200 FSQ 201 ReverTra Ace qPCR RT Master Mix PCR cDNA DNA ReverTra Ace qPCR RT Master Mix 200 FSQ 30 with gDNA Remover 10ul http www toyobo co jp bio 25 530 8230 2 8 TEL 06 6348 3786 FAX 06 6348 3833 E mail order lifescience toyobo jp
39. ReverTra Ace qPCR RT Kit Code FSQ 101 1 RNA RNA 65 C 5 RNA 5 x RT Buffer RT Enzyme Mix RNA 2 Nuclease free VWater Ll 5xRT Buffer 2 HI RT Enzyme Mix 0 5 HI Primer Mix 0 5 Hl RNA 0 5pg 1pg Total volume 10 UI 3 976 6 mmomnwnwa SCS GR 4 C hold 4 C 20 C PCR 2 9
40. ROX 5 KOD exo DNA polymerase KOD SYBR Green 1 2kb PCR 500bp 2kb KOD FX KOD Plus PCR 2kb
41. ROX reference dye Applied Biosystems 7000 7300 7700 7900HT StepOne 1x 150 StepOnePlus M 0 1x 1 500 Applied Biosystems 7500 7500Fast Agilent Technologies Roche Bio Rad BioFlux LineGene ROX reference dye 50 x ROX reference dye KOD SYBR qPCR Mix ROX 1 x qPCR Mix 50 x ROX 1 67ml 66 8ul QKD 201 1ml 40ul QKD 201T ROX 0 1 x qPCR Mix 50 x ROX 1 67ml 6 7ul QKD 201 1ml 4pl QKD 201T 6 2 PCR a PCR PCR b PCR 2 98 C 10 PCR 60 C 10 40 cycles 68 C 30 500bp 500bp 30 Data Collection
42. 1 Collect Data 1 Open Run hiethod Save Run Method Revert to Defaults Holding Stage 9 Cycling Stage hielt Curye Stage NumhernfCycles HH CO Continuous Step and Hold Enable AutoDelta Starting CyCle 1on 8 H C 98 0 C 5 H C 7 02 00 100 nO 10 IN WN 0015 N NM 2 N n0 1 1R0 C N 68 0 C N a 3 Nsnare eoo c xy 50 1 00 5D 0 00 ld II 1n 1 n 3 Step 1 gtep 1 Step 2 Step 3 Step 1 gten2 a Step 3 1 Sample Volume uL 2 2 step Add Step 3 step 3 4 GC 99 C 4 3 2 Applied Biosystems 7500 Fast Real Time System 1 4
43. 70 Pe THUNDERBIRD SYBR qPCR Mix 14 6 IT KOD SYBR qPCR Mix 2kb Tm Tm PCR PCR 3 C 5 C Tm A B A 4 k 0 oh op 4 95 60 i a a Tm em A 7 5 2 29 B Tm 199 82 82 357 84 85 C Tm Tm Ee
44. 98 C 10 60 C 10 68 C 30 1 Vortex 40 cycles 12 000rpm 5 optional 20ul 0 5ul lt 2ul ABI 7500 Fast Real Time System 1 Reverse 1 3 100bp Primer 8 _ Tm 79 C K K Wild type WT a wTkl Knock in KI A 90 Pri EN 341bp i Tm 84 C L Tem ae Cy 1 7 ASP PCR SNP 5 GC GC Tm 3 5 C
45. DNA PCR yeast RNA 3 plasmid DNA 1ug plasmid DNA 9 1 x 10 plasmid DNA kb d PCR 20ul 1 10 2Lul 1 3 1 2cm 200ul 5ul 10 cells
46. 0 Hl PCR 50ul 20pul XH X ul KOD SYBR qPCR Mix 25 ul 10 ul 1x Forward Primer 10 pmol 4 pmol 0 2 uM Reverse Primer 10 pmol 4 pmol 0 2 uM 50 x ROX reference dye 170 1 Hl 0 4 0 04ul 1x 70 1x DNA Yu Yu 50 Ll 20 ul 1 0 05 1 0 uM 2 Applied Biosystems Agilent Technologies ROX reference dye 1
47. 60bp 18 4 ALDH2 SNP DNA ALDH2 2 SNP SNP GC tail Tm Primer Gctai ALDH2 SNP ai G 487Glul 5 ctgcaggcatacact GAA gtgaaaactgtga 3 487Lys 5 ctgcaggcatacact AAA gtgaaaactgtga 3 Primer Prin 3 A 5 GTACGGGCTGCAGGCATAC 3 G 5 GCCGCCCTGCCCGCCACACTCACAGTTTTCACTIGEA 3 57bp A 5 CCCACACTCACAGTTTTCACTAITA 3 45bp GC tail C or T SNP IGlorlA PCR 98 C 2 98 C 10 60 C 10 68 C 30 50 cycles DNA
48. TOYOBO ie ldeas amp Chemistry KOD SYBR qPCR Mix Code No QKD 201 QKD 201T TOYOBO CO LTD Life Science Department OSAKA JAPAN A4642K 1 2 uu 2 3 4 6 5 I GC 6 I 7 ASP PCR SNP 8 RNA cDNA 9 10 S SYBR Molecular Probes Inc 1 KOD SYBR qPCR Mix SYBR Green PCR 2 x
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