クローニング実験ハンドブック
Contents
1. 201 1 7 1 TEL03 3271 8553 FAX 03 3271 7282 TEL077 543 7297 FAX 077 543 7293 TaKaRa TEL 077 543 6116 FAX 077 543 1977 http wwwr takara bio cojp http clontech takara bio coJp 2011 7 30K2. TA In Fusion cDNA N IdfdHa DNA TA cloning In Fusion DNA RNA mRNA cDNA PCR DNA DNA
3. DNA E coli HSTO8 Premium Competent Cells 9128 a F We Hind ll 10 000 U 1060A 6 500 BamH 10 000 U 1010A 6 500 Alkaline Phosphatase coli C75 50 U 2120A 10 500 Alkaline Phosphatase Calf intestine 1 000 U 2250A 10 500 PCR TaKaRa Ex 7gg Hot Start Version 250 U RRO006A 30 000 DNA Ligation Kit Mighty Mix 1 Kit 6023 26 000 TaKaRa DNA Ligation Kit LONG 1 Kit 6024 26 000 E coli HST08 Premium Competent Cells 1 Set 100 ul x 10 9128 21 000 E coli JM109 Competent Cells 1 Set 100 ul x 10 9052 19 000 DNA NucleoSpin Extract II 10 740609 10 5 300 NucleoSpin Plasmid QuickPure 10 740615 10 4 200 Agarose LO03 TAKARA 100g 5003 17 000 100 bp DNA Ladder 500 gl 100 3407A 19 000 A Hind Ill digest 100 ug 3403 8 500 4 5 7ag DNA P
4. _ 500 bp 3 NuSieve 3 1 2 0 1 10 10 x Loading Buffer Agarose L03 DNA S000 0 6 DNA 3 23 130 _1 soo 9 4900 a 899 8882 0 3 2 4 re EtBr SYBR Green EDNA Sdd
5. 1 1 20 pmol Alkaline Phosphatase BAP 0 3 0 6 U 10xBAP Buffer 5 ul up to 50 gl 1 37 65C 30 25 24 1 2 24 1 1 TE Buffer 20 ll DNA B 5 CIAP Calf Intestinal Alkaline Phosphatase 3 BAP Bacterial Alkaline Phosphatase 3 DNA DNA Ligation Kit Mighty Mix DNA A DNA B 25 250 fmol 50 ng 25 fmol 7 5 ul up to 15 ul Ligation Mix 16 C 30 25C 5 4
6. TA In Fusion DNA cDNA PCR PCR pk2J DNA pk2jpN dA DNA dA 5 _ total RNA In Fusion mRNA cDNA PCR 1
7. DNA DNA DNA TA 3 QA PCR dA 3 dT In Fusion 15 PCH PCR BamH 5 GATC EE Hind ll
8. DNA DNA 1 NucleoSpin Extract DNA 10 DNA 2 T Mighty TA cloning Kit 3ul pMD20 T Vector iul 50 ng PCR DNA i Ligation Mighty Mix 5 ll 1 16C 30 3 E coli HST08 Premium Competent Cells HST08 100 14 ml 10 jl 30 42C 45 1 2 37 SOC 1 ml
9. DNA DNA DNA 60 65 C 2 3 Ligation Kit DNA Q2 A2 TaKaRa DNA Ligation Kit LONG 6024 DNA 10 kb DNA 10 kb
10. 5 X In Fusion HD Enzyme Premix In Fusion 15 PCR DNA ein vitro Translation 2 3 DNA T4 DNA DNA Ligation Kit 1 DNA DNA a DNA
11. DNA PCH Advantage HD Polymerase Mix 5 x Advantage HD Buffer Mg 5 ul dNTP Mixture 2 5 mM each 2ul FW Primer RV Primer 0 2 0 3 uM Advantage HD Polymerase 2 5 units ul O25 Template 200 ng Sterile deionized HzO up to 25 ul 98C 10 sec 55C 5 sec 15 sec 30 cycles 72C 1 min kb In Fusion In Fusion Primer Design Tool http bioinfo clontech com infusion 2 PCR DNA NucleoSpin Extract Cloning Enhancer PCR ST Cloning Enhancer 2 ul 37C 15 80C 15 1 DNA B 3 In Fusion Cloning 5 Xx
12. EE ET 50 740588 50 10 400 NUM69OSBIOP SI 250 740588 250 48 400 DNA 50 740615 50 10 500 NucleoSpin Plasmid QuickPure 250 740615 250 35 700 10 740410 10 13 700 DNA NucleoBond aa Midi 50 740410 50 60 000 PCH a 50 740609 50 12 300 0 1 250 740609 250 41 000 total RNA NucleoSpin RNA II 50 740955 50 31 800 10 DNA DNA 1 1 2 5 1 150 ml
13. StellarTM Competent Cells DNA pUC o X Gal 8 9 RNA RT PCR Library Reverse Transcriptate RTase cDNA amp cDNA MMLV RTase 1st Strand cDNA avian myeloblastosis virus AMV moloney murine leukemia virus M MLV 2 mRNA
14. 30C 10 min 3 42C 50C 4 30 60 min 1st strand 8 NR PrimeScript Il 1st strand cDNA Synthesis Kit 50 6210A 34 000 PrimeScript RT PCR Kit 50 RRO14A 31 000 RT PCR PrimeScript One Step RT PCR Kit Ver 2 50 RRO55A 42 000 cDNA SMARTer PCR cDNA Synthesis Kit 10 634925 174 000 PrimeScript Reverse Transcriptase 10 000 U 2680A 29 000 PCR DNA DNA DNA EE NucleoSpin Plasmid QuickPure 1 3 ml
15. Hind Ba77H double digestion DNA 1 ug Hind ll ud BamH 1 10xK Buffer up to 20 pl 37C 1 10 Loading Buffer DNA DNA UV NucleoSpin Extract Il DNA 10 DNA A b PCR 5 DNA PCH 5 3 TaKaRa Ex 7ag Hot Start Version PCR 10 x Ex 7gg Buffer Mg plus 5ul dNTP Mixture 2 5 mM 4 200 uM primer 1 0 2 1 0 uM fnal conc primer 2 0 2 1 0 uM fnal conc DNA lt 500 ng TaKaRa Ex 7gg HS 25 up to 50 uil
16. 6 7 In Fusion Clontech In Fusion DNA 15 In Fusion contecn 50 bp 15 kb 1 DNA In Fusion 15 a 2 IFusion HD Enzyme amp dHeO Pin Fusion DNA 15 15
17. 98C 10 sec 55C 30 sec 30 cycles 72C 1 min kb 1 2 Hind ll A BamH 1 10XK Buffer 2 up to 20 ul 37C 1 1 NucleoSpin Extract Il DNA 10 i DNA A 2 DNA 1 g Hind ll Wal BamH Wl 10xK Buffer ll up to 20 pul 37 1 NucleiSpin Extract DNA 10 TE Buffer 20 Al DNA B 1 DNA
18. 37C 1 160 225 rpm LB LB Amp X Gal 37C coli JM109 X Gal IPTG LB 37C 4 PCR EmeraldAmp PCR Master Mix EmeraldAmp PCR Master Mix 2 Xx Premix 0 2 uM final conc 0 2 uM final conc up to 50 Forward Primer Reverse Primer dHzO PCR 98C 10 sec 55C 30 sec 72C 1 min kb 30 cycles PCR 5 LB Amp 2 ml 1 NucleoSpin Plasmid QuickPure DNA 10 D
19. 3 ee 2150 ml 2 3 NN BR 3 3 holder 50 60
20. AMV RTase MMLV PrimeScript RTase cDNA RNA 3 1 dT mRNA polyA tail 3 CDNA 2 6 59 mRNA CDNA 3 cDNA 1st strand cDNA A a 70C 15 o PrimeScripte RTase RNA Primer 1st strand CDNA PCR 2nd strand cDNA Oligo dT primer 50 pmol or Random primer 6mers 50 pmol or Gene specific primer 2 pmol dNTP mixture 10 mM each
21. 100 mg NT 200 50C10 NucleoSpin 11 000 Xx g 1 NT3 EtOH 700 11 000 x g 1 11 000 Xx g 2 NE 15 lt 50 ul 1 11 000 x g 1 NucleoSpin Extract Il PCR NucleoSpin Binding Buffer Elution Buffer DNA
22. TaKaRa Ex 7ag PrimeSTAR TaKaRa LA 7agg TaKaRa Taq MigythAmp EmeraldAmp SapphireAmp SpeedSTAR PrimeSTAR o PC Mighty Cloning Reagent Set Blunt End Ori lacZ T Vector pMD20 2 736 bp Multicloning site _ Mighty TA cloning Kit 20 6028 21 000 TA Mighty TA cloning Reagent Set for PrimeSTAR 20 6019 32 000 T T Vector pMD20 1 g 3270 9 500 T Vector pMD19 Simple 1ug 3271 9 500 E coli HST08 Premium Competent Cells 1 Set 100 ul x 10 9128 21 000 E coli JM109 Competent Cells 1 Set 100 ul x 10 9052 19 000 E coli DH5a Competent Cells 1 Set 100 ul X 10 9057 19 000 PCR EmeraldAmp PCR Master Mix 160 RR300A 16 000 DNA NucleoSpin Extract II 10 740609 10 5 300 NucleoSpin Plasmid QuickPure 10 740615 10 4 200 Agarose L03 TAKARA 100g 5003 17 000 100 bp DNA Ladder Dye Plus 500 100 3422A 19 000 A Hind Ill digest 100 ug 3403 8 500
23. 1 Agarose L03 8 125 A Hind Ill digest 100 bp 1 5 SeaKem GTG 2 Agarose L03 TAKARA 100g 5003 17 000 NuSieve 3 1 Agarose 125g 50090 62 700 AccuGENEW 10 x TAE Tris Acetate EDTA Buffer 1L 50844 8 500 Mupid 2plus lt M 2P 40 762 100 bp DNA Ladder Dye Plus 500 100 3422A 19 000 1 kb DNA Ladder Dye Plus 500 100 3426A 13 000 A Hind lll digest 100 ug 3403 8 500 Mupid Scope WD MS WD 340 953 1 Lonza 2 Hot Start PCR Licensed under U S Patent No 5 338 67 1 and 5 587 287 and corresponding patents in other countries In Fusion Cloning Products This product is covered by U S Patent No 7 575 860 and European Patent No EP1741787
24. E coli HST08 Premium Competent Cells HST08 100 gull 14 ml 10 30 42C 45 1 2 37 SOC 1 ml 37 1 160 225 rpm LB 37 5 PCR 6 PCR 5 10 Q1 A1 DNA
25. Y 10 639633 22 000 In Fusion HD Cloning Kit w NucleoSpin Y 10 639639 24 000 In Fusion HD Cloning Kit w Competent Cells Y 10 639642 36 000 In Fusion HD Cloning System CE Y Y 10 639636 37 000 In Fusion HD Cloning System Y vv 10 639645 39 000 In Fusion HD Cloning Kit 10 639648 21 000 In Fusion HD EcoDryW Cloning Kit 8 639689 21 000 In Fusion HD 50 100 In Fusion HD EcoDry Cloning Kit In Fusion HD 24 8 x3 96 96 In Fusion HD Cloning Enhancer PCH In Fusion PCR dNTP In Fusion NucleoSpin Extractl PCR
26. PCR PCR PCH In Fusion Cloning Enhancer 50 15 In Fusion 1 DNA PCR A 1 DNA PCHR In Fusion Primer DNA Forward Primer 5 NNNNNNNNNNNNNNN gt 8 5 NNNNNNNNNNN INNNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNN INNNNNNNNNNN 5 NNNNNNNNNNNNNNN
27. 1 ul D s total RNA 5 g lt ER DD i i 1 cDNA RNA RNase RNase 65C 5 2 RNA RNase 20 RNA Primer Mixture 10 ul 2 RNA 5 X PrimeScript Buffer 4 GSciininDiton US 3 Random 6 mers PrimeScript Reverse Transcriptase 100 200 units EY 0 4 RNA MMLV RTase PrimeScript RTase 42 RNA
28. 11 000 Xx g 30 A1 250 Vortex A2 250 7 8 5 A3 300 ll 7 8 1 1 000 Xx g 5 NucleoSpin 11 000 Xx g 1 AQ EtOH 450 11 000 Xx g 3 AE 50 ul 1 11 000 x g 1 NucleoBond pH pH DNA DNA NucleoSpin Extract
29. 5 Forward Primer Reverse Primer 5 NNNNNNNNNNNNNNN gt 5 NNNNNNNNNNNNNNN INNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNN INNNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN 5 Forward Primer Reverse Primer 5 NNNNNNNNNNNNNNN gt 5 NNNNNNNNNNNNNNNNNNNINNNNNNNNNNNNNNN 3 3 3 NNNNNNNNNNNNNNNINNNNNNNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN 5 Reverse Primer 15 In Fusion DNA 15 PCH 15 5 In Fusion Primer In Fusion Cloning A overhang PCH A overhang
30. PCR PCR DNA 2 7gg DNA Polymerase Pol family A Pfu DNA Polymerase o family B Pol 1 DNA Pol Il PCR TaKaRa Ex 7gg dA PCH TA ag DNA 3 5 exonuclease TA PCR TA 3 QA TA Mighty TA Cloning Kit g DNA TA D TA Cloning Kit 2 T T V
31. In Fusion HD Enzyme Premix 2ul A x ul CE PCR B y dHzO up to 10 gl 50C 15 In Fusion 4 StellarrM Competent Cells 636763 coli HSTO08 Premium Competent Cells 9128 1x108 cfu ug DNA 5 PCR PCR 5 10 D DNA 415 7 10 Fragment 2 Fragment 1 gt Fragment 3 In Fusion Cloning 1 kb DNA In Fusion HD Cloning Kit PCR 10 7 Lo In Fusion HD Cloning Kit w Cloning Enhancer
32. ector pMD20 T Vector pMD19 Simple T Vector pMD19 Simple pUC19 D 5 kb PCR PCR 5 kb TA Mighty Cloning Reagent Set Blunt End 6027 TA Mighty TA cloning Reagent Set for PrimeSTAR PCR 3 dgA A overhang mixture TA PCR Pol lI a family A family B dA TA 3
33. olymerase PCR 3 dA TA 3 dgT T PCR dA DNA 1 DNA DNA PCR TaKaRa Ex 7gg 10 x Ex Taqg Buffer Mg plus 5ul dNTP Mixture 2 5 mM 4 ul 200 uM primer 1 0 2 1 uM final conc primer 2 0 2 1 uM final conc DNA lt 500 ng TaKaRa Ex Tag 2 up to 50 ul 98C 10 sec 55C 30 sec 72C 1 min kb 30 cycles PrimeSTAR o PCH 3 QA 2 T
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