クローニング実験ハンドブック
Contents
1. 2014 4 2015H 4HHHHHHHHH TA In Fusion ns cDNA that s Go0 ccience Tahaia CCiontech http www takara bio co jp DNA TA In Fusion DNA RNA mRNA2. DNA Q2 A2 TaKaRa DNA Ligation Kit LONG 6024 DNA 10 kb DNA 10 kb DNA E coli HST08 Premium Competent Cells 9128 Takara Cut Site Navigator DNA Hind 1 10 000 U 1060A 6 700
3. Cloning Enhancer 50 15 In Fusion 1 DNA lt DNA PCR PrimeSTAR Max DNA Polymerase PCR A PrimeSTAR Max Premix 2 25 Forward Primer 0 2 0 3 uM DNA PCR In Fusion Primer ReverSe Primer 0 2 0 3 uM DNA 2 CE a Sterile deionized H20 up to 50 pl 5 NNNNNNNNNNNNNNN gt 5 NNNNNNNNNNN NNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNNI NNNNNNNNNNN 5 99 10 sec NN NN 55 5 sec 15 sec 30 35 c
4. DNAO P 1 DNA DNA PCR TaKaRa Ex 7gg 10 x Ex Tag Buffer Mg plus 5 ul dNTP Mixture 2 5 mM 4 Al 200 uM Forward Primer 0 2 1 uM fnal conc Reverse Primer 0 2 1 uM fnal conc DNA lt 500 ng TaKaRa Ex Tag 251U up to 50 gl 98C 10 sec 55C 30 sec 72C 1 min kb 30 cycles PrimeSTAR o PCR 3 dA DNA NucleoSpin Gel and PCR Clean up PCR DNA 10 DNA DNA DNA
5. avian myeloblastosis virus AMV moloney murine leukemia virus M MLV 2 mRNA AMV RTase MMLV PrimeScript cDNA RNA 3 1 dT mRNA bpolyA tail 3 cDNA 2 6 9 mRNA rRNA RNA cDNA 3 cDNA 1st strand cDNA PrimeScript Il 1st strand cDNA Synthesis Kit D cDNA
6. RNA Primer cDNA Oligo dT Primer 50 uM fg or Random 6mers 50 uM I or Gene Specific Primer 2 pmol dNTP mixture 10 mM each 1 ul RNA total RNA 25 pg RNA or polyA RNA 1 ug RNase free dHzO up to 10 ul 8 NNN 65C 5 cDNA RNA RTase 20 RNA W ESBEITE PrimeScript II RTase 5 PrimeScript RNase Inhibitor 40 U ul 0 5 ul PrimeScript II RTase 200 U l 1 ul RNase free dHzO up to 20 ul RNA 42C RNA RTase RNA
7. 30C 10 min Random 6mers 42C 30 60 min 95C 5 1st strand cDNA PrimeScript Il 1st strand cDNA Synthesis Kit 35 000 7 2 PCR PrimeScript Il High Fidelity RT PCR Kit 50 000 RT PCR primescript Il High Fidelity One Step RT PCR Kit 50 000 9 TakaRa contech PCR DNA DNA DNA DNA NucleoSpin Plasmid EasyPure 2 10 ml 12 000 x g 30
8. DNA NucleoSpin Plasmid DNA NucleoSpin Plasmid EasyPure DNA NucleoSpin Gel and PCR Clean up 100 mg NTI 200 ul 50 C10 NucleoSpin 11 000 x g 30 NT3 EtOH 700 gl 11 000 x g 30 NE 15 30 ul 1 11 000 x g 1 EMH1 000 x g 1 NucleoSpin Gel and PCR Clean up PCR PC 100 OO NTI 200 ul NucleoSpin
9. 3 holder 50 60 _ 500 bp PrimeGel Agarose PCR Sieve 3 1 10 10 x Loading Buffer Nit 0 i 5 000 bp PrimeGel Agarose LE 3 40K 0 5 500 5 000 bp Agarose L03 PrimeGel M Agarose LE 1 20K 1 DNA
10. 3 css 1 i 1 0o0 3 TN 6oo 10 4472 Ln 1 kb DNA Ladder 5 2 690 AOSSDO08B a 4 RN 2 A EooT14 1 digest 5 4 1 PrimeGel Agarose LE 1 20K GAT 922 EtBr SYBR Green a i i bp bp Agarose L03 TAKARAI 1 kb 5003 17 000 primeGeI Agarose LE 1 20K GAT 1 kb 100g 34 000 PrimeGel Agarose PCR Sieve PCR 1 kb 100 g 48 000 Tris Acetate EDTA Buffer TAE 50 x Powder DH8 3 1 1 L 50 x TAE 1 16 000 Tris Borate EDTA Buffer TBE Powder pH8 3 1 1 L TBE 10 10 000 100 bp DNA Ladder Dye Plus 100 1 500 bp 11 500 100
11. DNA 10 DNA A 2 DNA 1 g Hind ll 1 ul BamH 1 ul 10xK Buffer 2 ul ph K 2 9 37C 1 NucleoSpin Gel and PCR Clean up DNA 10 DNA B 1 DNA 1 20 pmol Alkaline Phosphatase BAP 0 3 0 6 U 10 x BAP Buffer 5 Ll up to 50 ul 25 24 1
12. DNA In Fusion dA PCR dA In Fusion TA EE BamH 5 GATC um me GA 5 Hind ll 5 x In Fusione HD In Fusion Enzyme Premix nN 15 2G PCR DNA DNA NucleoSpin Plasmid EmeraldAmp MAX PCR Master Mix EasyPure 77 vitro Translation 1 TakaRa contech DNA
13. DNA DNA 4 3 dA PCR dA 3 dT 15 In Fusion PCR PCH gt DNALigation Kit Mighty Mix P4 BamH Hind Il a Mighty Cloning Reag
14. cDNA PCR DNA DNA TA In Fusion DNA cDNA PCR PCR SW DNA cDNA DNA mMm DNA cDNA PCR DNA DNA
15. A1 RNase A 150 gl Vortex A2 250 5 2 A3 350 12 000 Xx g 3 NucleoSpin Plasmid EasyPure 1 000 2 000 x g 30 AQ EtOH 450 ul 12 000 x g 1 AE 50 ul 1 12 000 x g 1 NucleoBond pH pH
16. Mighty Cloning Reagent Set Blunt End 6027 Multicloning site Mighty TA cloning Kit 22 000 Mighty TA cloning Reagent Set for PrimeSTAR 33 000 TA T T Vector pMD20 10 000 T Vector pMD19 Simple 10 000 20 1 Set 100 pul x 10 Mighty Cloning Reagent Set Blunt End 30 000 coli HST08 Premium Competent Cells 22 000 E coli JM109 Competent Cells 1 Set 100 ul Xx 10 20 000 E coli DH5o Competent Cells 1 Set 100 ul Xx 10 20 000 PCR EmeraldAmp MAX PCR Master Mix RR320A DNA 740609 10 NucleoSpin Gel and PCR Clean up NucleoSpin Plasmid EasyPure 740727 10 Agarose L03 TAKARA 17 000 100 bp DNA Ladder Dye Plus 500 100 100 ug 19 000 A Hind digest 8 500 PCR TakKaRa Ciontech 6 Clontech In Fusion
17. Binding Buffer Elution Buffer DNA 74072 50 10 500 74072 250 36 00 NucleoBond Xtra Midi 740410 10 13 700 DNA EF NucleoBond Xtra Midi EF 740420 10 21 900 PCR total RNA NucleoSpin RNA NucleoSpin Gel and PCR Clean up 740609 50 12 500 740609 250 45 000 740955 50 33 500 TakaRa clontech 10 DNA
18. DNA 10 DNA A b PCR 5 DNA PCH 5 3 Tks Gflex DNA Polymerase PCR 2 x Gflex PCR Buffer Mg21 dNTP plus 25 ul 0 2 0 3 uM fnal conc 0 2 0 3 uM fnal conc 500 ng 1 25 U up to 50 ul Forward Primer Reverse Primer DNA Tks Gflex DNA Polymerase 94C 1 min 98C 10 sec 60 0 15 sec 68C 30 sec kb PCR NucleoSpin Gel and PCR Clean up 10 30 cycles 3 TakaRa contech PCR 2 ul Hind I 1 ul BamH 1 ul 10xK Buffer 2 ul up to 20 37 C 1 NucleoSpin Gel and PCR Clean up
19. Ni Alkaline Phosphatase E coli C75 Alkaline Phosphatase Calf intestine 10 000 U 1010A 6 700 11 000 11 000 50 U 2120A 1 000 U 2250A Tks Gflex DNA Polymerase 250 U RO60A 31 500 DNA Ligation Kit Mighty Mix TaKaRa DNA Ligation Kit LONG E coli HSTO8 Premium Competent Cells 1 Kit 6023 2 000 2 000 22 000 1 Kit 6024 1 Set 100 ul Xx 10 9128 coli JM109 Competent Cells 1 Set 100 ul Xx 10 9052 20 000 DNA NucleoSpin Gel and PCR Clean uUD NucleoSpin Plasmid EasyPure 10 740609 10 G00 10 740727 10 4 200 Agarose LO3 TAKARA 100g 5003 17 000 100 bp DNA Ladder A Hind ll digest 500 100 3407A 19 000 TaKaRa Clontech 4 Tagqg DNA Polymerase PCRH 3 dA TA 3 TA dT T PCR dA
20. DNA 15 F 1 FUSION _ 50 bp 15 kb 1 DNA In Fusion 15 m5 x In Fusion HD E amp dH20 DIn Fusion DNA 15 15 PCR PCR PCHRH In Fusion
21. PCR 6 PCR 5 10 DD Takara Cut Site Navigator Q1 A1 DNA DNA DNA DNA 60 65 2 3 Ligation Kit
22. 3 dA A overhang mixture DNA Polymerase Pol family A Pfu DNA TA Polymerase o family B Pol 1 DNA Pol 1 PCR 7gKgg Ex 7gg 3 qdA PCR TA o DNA 3 5 exonuclease TA PCR TA 3 dA TA Mighty TA cloning Kit o DNA TA D T TA cloning Kit
23. NucleoSpin Gel and PCR Clean up 2 T Mighty TA cloning Kit 3 ul pMD20 T Vector 1 50 ng PCR 1 ul DNA Ligation Mighty Mix 5 ll 16C 30 5 IMdH4 contech 3 E coli HST08 Premium Competent Cells HST08 100 ul 14 ml ee 0 30 426 1 2 oe ml 37 1 2 160 225 rpm 2 37 C coli JM109 X Gal IPTG LB 37 C 4 PCR EmeraldAmp
24. DNA 1 1 150 ml 1 5 g 2150 ml 1 2 5 3 2 3 10 3
25. 2 T T Vector pMD20 T Vector pMD19 Simple Or T Vector pMD19 Simple pUC19 Pol 1 family A dA TA q family B 3 PrimeSTAR Tks Gflex TaKaRa Ex Tag TaKaRa LA Tag TaKaRa Tag MigythAmp EmeraldAmp MAX SapphireAmp SpeedSTAR PrimeSTAR o PCR Mighty Cloning Reagent Set Blunt End lacZ T Vector pMD20 D 5 kb PCR 2 736 bp PCR 5 kb TA
26. In Fusion5 HD Stellar Competent Cells DNA pUC o X Gal CloneAmp HiFi PCR Premix PCR In Fusion DNA PCR In Fusion HD Cloning Plus TakKaRa Clontech 8 RNA RT PCR Library amp cDN A Reverse Transcriptate RTase cDNA PrimeScript Il 1st strand cDNA Synthesis Kit cDNA
27. 19 000 1 kb DNA Ladder Dye Plus 1 10 kb 10 500 100 3426A 13 000 Mupid 2plus 40 760 Mupid exU EXU 1 56 950 In Fusion Cloning Products This product is covered by U S Patent No 7 575 860 and European Patent No EP1741787
28. 2 24 1 1 TE Buffer 20 ul DNA B 5 CIAP Calf Intestinal Alkaline Phosphatase 3 BAP Bacterial Alkaline Phosphatase 3765C 30 3 DNA DNA Ligation Kit Mighty Mix DNA A DNA B 25 250 fmol 50 ng 25 fmol Ligation Mix SV up to 15 ul 16C 30 25 C 5 4 E coli HST08 Premium Competent Cells HST08 100 14 ml oe 0 30 426 a 1 2 ee ml 37 C 1 160 225 rpm LB 37 5
29. 2014 4 1 i TEL03 3271 8553 FAX 03 3271 7282 TEL077 565 6969 FAX 077 565 6995 TaKaRa TEL 077 543 6116 FAX 077 543 1977 Website http www takara Dio coJp Facebook http www facebook com takarabioJp 2014 3 36K
30. A T4 DNA DNA Ligation Kit 1 DNA DNA a DNA Takara Cut Site Navigator Hind Ba77H double digestion DNA 1 g Hind 1 ul BamH 1 ul 10xK Buffer 2 Up to 201uIR 37 1 10 Loading Buffer DNA DNA UV NucleoSpin Gel and PCR Clean up
31. In Fusion Cloning A overhang PCHR A overhang In Fusion Primer 7 TakaRa contech 2 PCR DNA NucleoSpin Gel and PCR Clean uD Cloning Enhancer PCR Cloning Enhancer 37C 15 80C 15 DNA B 3 In Fusion Cloning 5 x In Fusion HD Enzyme Premix 2 ul A x Ul CE PCR B y ul dHzO up to 10 ul 50C 15 10 EcoDryW 8 4 StellarTM Competent Cells 636763 E coli HSTO08 Premi
32. MAX PCR Master Mix EmeraldAmp MAX PCR Master Mix 2XPremix 25 ul 0 2 uM final conc 0 2 uM final conc up to 50 ul Forward Primer Reverse Primer PCR 98C 10 sec 60C 30 sec 72C 1 min kb PCR 30 cycles 5 LB Amp 2 ml NucleoSpin Plasmid EasyPure DNA 10 DNA lt D PCR PCR DNA 2 7g9 Mighty TA cloning Reagent Set for PrimeSTAR PCR
33. ent Set 9 2 Blunt End TaKaRa DNA Ligation Kit P4 LONG TA P6 A dA pg In Fusion 15 DNA DNA OK In Fusion HD DNA RNA PrimeScript PCR Enzymes Guide Cloning Kit Clean uD TakaRa Clontech 2 DN
34. r 639633 24 000 In Fusion HD Cloning Kit w NucleoSpin 639639 26 000 In Fusion HD EcoDry Cloning Kit 639689 23 000 In Fusion HD Cloning Plus 638909 47 400 KEI NN In Fusion HD Cloning Plus CE 638916 45 300 In FusionB HD 50 100 In Fusion HD EcoDry Cloning Kit 24 8 X 3 96 96 In Fusion HD Cloning Enhancer PCR In Fusion PCR dNTP In Fusion NucleoSpin Gel and PCR Clean up PCR E
35. um Competent Cells 9128 1x109 cfu ug DNA 5 PCR PCR 5 10 D DNA 415 7 10 Fragment 2 Fragment 1 Fragment 3 In Fusion Cloning 1 kb DNA In Fusion HD Cloning Kit PCRH 10 7 Steller CloneAm Cloning Competent HiFi PCR Enhancer NucleoSpin Cell PCR PrimeSTAR In Fusion HD Cloning Kit 639648 23 000 In Fusion HD Cloning Kit w Cloning Enhance
36. ycles Td Reverse Primer 72C5 sec kb Sr NNNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN NNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNN INNNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN 5 Forward Primer Reverse Primer Sr NNNNNNNNNNNNNNN a 5 NNNNNNNNNNNNNNNNNNNINNNNNNNNNNNNNNN 3 3 3 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN 5 Reverse Primer In Fusion http www takara DiO CO jD infusion tool 15 In Fusion DNA 15 PCR 15 5 In Fusion Primer
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