『KOD -Plus- Mutagenesis Kit』を用いたルシフェラーゼ 遺伝子へのHis
Contents
1. 17 7 Amax 1 No INo 2 1 GGG Gly 9 33 TCT Ser 1 no 58S 2 eoc Ala 34 AcCA Thr Amax nm 3 GmT Val 35 Acc Thr ac sh 4 eeT Gly 36 ACG Thr LM eS 5 ATG Met 37 ACT Thr se 6 GTA Val 38 TAT Tyr aks 7 erTe Val 39 eTc Val 3 8 ecT Ala 3 40 CGA Arg ND 9 CeT Arg 41 cec Arg 4 10 CAT His 3 42 AAcC Asn 0 ND 11 CTT Leu 3 43 GAT Asp S i ND 12 ece Ala 2 44 Tec Cys Me ao NM 13 AeA Arg 2 45 CAA GIn Ala S60 14 GAC Asp 46 CAG GIn 9 i a Sa 15 eec Gly 2 47 ATA lle 12. 4 6 co OT co 1 CATCACCATCACCATCAC 5 Primer 1 primer 55mer Primer PCR p DH5 Code No DNA 903 co 1 4 Emerald Luc His tag CGGATAACAATTTCACAOAGGAAACAGACCATGGAGAGAGAGAAGAACGTGGTGTACGGCCCCGAGCCCAAG 3 GCCTATTGTTAAAGTGT CGGATAACAATTTCACACAGGAAACAGACCATG GCCTATTG AAAGIGIGI6GIMMGI6IGGIAC 20 6 a CGGATAACAATTTCACACAGGAAACAGACCATG GCCTATTG AAAGIGIGI6CIINGI6IGGIA6 Emerald Luc CDS SD NMWetGluArgGluLysAsnValValTyrGIyProGluProLys TGTCTGGTACCTCTCTCTCTTCTTGCACCACATGCCGGGGCTCGGGTTC 5 5 Primer 1 3 GGA GAA OARGGNGGNG TACGGCCCCGAGCCCAAG 3 C
3. PCR nd site mutation 3 KOD Plus Mutagenesis Kit His tag Sm Ei 1 Template plasmid Inverse PCR 1 KOD Plus Mutagenesis KIt w PCR Primer x Inverse PCR 5 10 0 5 2hr gt Dpn 1 Plasmid lt 37 C 1hr gt PCR Self ligation Kinase Ligase 16 C 1hr gt Transform E coli 3 KOD Plus MutagenesiS Kit Inverse PCR Emerald Luc plasmid
4. p1 3 T Nakatsu Nature 2006 Mar 16 440 7082 372 6 Emerald Luc 882 lle 22 N LeuLysAlalleGlnAspTyrGluValArgSerVallleAsnValProSerThrlleLeuPheLeuSerLys 6 8 lle 5 CTGAAGGCCATCCAGGACTACGAGGTGAGGAGCGTGATCAACGTTCCCTCCACAATCCTGTTCCTGTCCAAG 3 NNN 3 4 3 GACTTCCGGTAGGTCCTGATGCTCCACTCCTCGCACTAGTTGCAAGGGAGGTGTTAGGACAAGGACAGGTTC 5 64 Primer 3 primer MM a 5 NNNAACGTTCCCTCCACAATCCTGTTCC 25mer Primer 4 primer 5 CTGAAGGCCATCCAGGACTACGAGGTGAGGAGCGTGATCAAGGMTCCCTECACAATECTGMNCCTGTCCAAG 3 3 GACTTCCGGTAGGTGCTGAMGCTCCACMCCTEGCAGTAGTTGCAAGGGAGGTGTTAGGACAAGGACAGGTTC 5
5. A B His tag His tag Ni T N His tag C N C Emerald Luc
6. 3 iQGICCIGAIGGTCCACICGTCGCA6G 5 DH5o Code No DNA 9093 Primer 282 LeuLysAlalleGInAspTyrGluValArgSerVal 2AsnValProSerThrlleLeuPheLeuSerLys CTGAAGGCCATCCAGGACTACGAGGTGAGGAGCGTGNNNAAGGTICCCI6GACAATCCIGTHGGTGTCCAAG GACTTCCGGTAGGTCCTGAIGGTGCACTCCTGGCACNNNTTGCAAGGGAGGTGTTAGGACAAGGACAGGTTC TH 6 Emerald Luc 96 1 GGG 9 39 GIn Lys 18 le 1 7 96 n
7. IKOD Plus Mutagenesis Kit His tgg Site directed mutagenesis Kunkel PCR 2nd site mutation KOD Plus Mutagenesis Kit 1 PCR KOD Plus Inverse PCR iPCR Inverse PCR bp 10bp 100bp
8. Mutagenesis Kit 20 SMK 101 38 000 KOD Plus 10x Buffer for iPCR 2mM dNTPs Dpnl T4 Polynucleotide Kinase Ligation high Control Plasmid Control Primer 1 Control Primer 2 UPLOAD
9. gi ls 16 AGC Ser 48 ATC lle 9 a 17 Tee Trp 2 49 CTA Leu Le IP NB 18 ecA Ala 50 CTC Leu 5 8 19 AGG Arg 51 TTA Leu 3 a a a a MM NB 21 AAT Asn 53 AAA Lys ae TGT Cys 54 AAG Lys 23 GAA Glu 55 TTT Phe 7 2 25 GeA Gly 57 ccT Pro 27 ATT lle 59 ToCC Ser 8 28 CTG Leu 60 TCG Ser 29 TTC Phe 61 TGA Stop 9 30 CCC Pro 62 TAA Stop 31 CCG Pro 63 TAG Stop 32 TCA Ser 64 TAC Tyr Primer 96 Wild I282V I282A gt cE gt 4 Relative Intensity 550 600 650 700 450 500 550 600 650 700 Wavelength nm Wavelength nm Amax 541nm 552nm 560nm 7 Val Ala 8 Val Ala N Na KOD Plus Mutagenesis Kit Inverse PCR tag 1 00Dp KOD Plus Mutagenesis Kit Code No EE KOD Plus
10. TCTCTCTCTTCTTGCACCACATGCCGGGGCTCGGGTTC 5 Primer 2 EL N MetHisHisHisHisHisHisGluArgGluLysAsnValVal GTAGTGGTAGTGGTAGTGCTCTCTCTCTTCTTGCACCAC 5 A SDS PAGE B 12 His tag ee His tag His tag plasmid His tag His tag plasmid MagExtractor His tag Code No NPK His tag plasmid His tag 701 ye SDS PAGE 5 His tag plasmid His tag Sharpline Low range markers EE Code No PMW 201 5 His tag ELuc His tag
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