Dam Ī G m ATC ī Ă Dcm Ī C m CWGG īȤɢɍ CpG Ī m CG īʹʙ˃ݛĪറȧī

ਊ೧ ༯ᇙ Dam Dcm CpG ^{ਊ೧ ༯ᇙ} Dam Dcm CpG

BsaAI YAC/GTR EarI CTCTTC(1/4) ol

BsaBI GATNN/NNATC ol scol EciI GGCGGA(11/9) scol

BsaHI GR/CGYC scol Eco53kI GAG/CTC scol

BsaJI C/CNNGG EcoNI CCTNN/NNNAGG

BsaWI W/CCGGW EcoO109I RG/GNCCY ol

BsaXI (9/12)ACNNNNNCTCC(10/7) EcoP15I CAGCAG(25/27)

BseRI GAGGAG(10/8) EcoRI G/AATTC scol

BseYI CCCAGC(-5/-1) ol EcoRI-HF G/AATTC scol

BsgI GTGCAG(16/14) EcoRI-HF RE-Mix G/AATTC scol

BsiEI CGRY/CG EcoRV GAT/ATC scol

BsiHKAI GWGCW/C EcoRV-HF GAT/ATC scol

BsiWI C/GTACG EcoRV-HF RE-MixGAT/ATC scol

BslI CCNNNNN/NNGG scol scol FatI /CATG

BsmI GAATGC(1/-1) FauI CCCGC(4/6)

BsmAI GTCTC (1/5) scol Fnu4HI GC/NGC ol

BsmBI CGTCTC(1/5) FokI GGATG(9/13) ol ol

BsmFI GGGAC(10/14) ol ol FseI GGCCGG/CC scol

BsoBI C/YCGRG FspI TGC/GCA

Bsp1286I GDGCH/C FspEI

BspCNI CTCAG(9/7) HaeII RGCGC/Y

BspDI AT/CGAT ol HaeIII GG/CC

BspEI T/CCGGA ol HgaI GACGC(5/10)

BspHI T/CATGA ol HhaI GCG/C

BspMI ACCTGC(4/8) HincII GTY/RAC scol

BspQI GCTCTTC(1/4) HindIII A/AGCTT

BsrI ACTGG(1/-1) HindIII-HF A/AGCTT

BsrBI CCGCTC(-3/-3) scol HinfI G/ANTC scol

BsrDI GCAATG(2/0) HinP1I G/CGC

BsrFI R/CCGGY HpaI GTT/AAC scol

BsrGI T/GTACA HpaII C/CGG

BssHII G/CGCGC HphI GGTGA(8/7) ol

BssKI /CCNGG ol ol Hpy99I CGWCG/

BssSI CACGAG(-5/-1) Hpy166II GTN/NAC ol

BstAPI GCANNNN/NTGC scol Hpy188I TCN/GA ol

BstBI TT/CGAA Hpy188III TC/NNGA ol ol

BstEII G/GTNACC HpyAV CCTTC(6/5) ol

BstEII-HF G/GTNACC HpyCH4III ACN/GT

BstEII-HF RE-Mix G/GTNACC HpyCH4IV A/CGT

BstNI CC/WGG HpyCH4V TG/CA

BstUI CG/CG KasI G/GCGCC

BstXI CCANNNNN/NTGG scol KpnI GGTAC/C

BstYI R/GATCY KpnI-HF GGTAC/C

BstZ17I GTA/TAC scol KpnI-HF RE-Mix GGTAC/C

Bsu36I CC/TNAGG LpnPI

BtgI C/CRYGG MboI /GATC ol

BtgZI GCGATG(10/14) MboII GAAGA(8/7) ol

BtsI GCAGTG(2/0) MfeI C/AATTG

BtsIMutI CAGTG(2/0) MfeI-HF C/AATTG

BtsCI GGATG(2/0) MfeI-HF RE-MixC/AATTG

Cac8I GCN/NGC scol MluI A/CGCGT

ClaI AT/CGAT ol MluCI /AATT

CspCI (11/13)CAANNNNNGTGG(12/10) MlyI GAGTC(5/5)

CviAII C/ATG MmeI TCCRAC(20/18) ol

CviKI-1 RG/CY MnlI CCTC(7/6)

CviQI G/TAC MscI TGG/CCA ol

DdeI C/TNAG MseI T/TAA

DpnI GA/TC ol MslI CAYNN/NNRTG

DpnII /GATC MspI C/CGG

DraI TTT/AAA MspA1I CMG/CKG ol

DraIII-HF CACNNN/GTG scol MspJI

DrdI GACNNNN/NNGTC scol MwoI GCNNNNN/NNGC scol

EaeI Y/GGCCR ol ol NaeI GCC/GGC

EagI C/GGCCG NarI GG/CGCC

EagI-HF C/GGCCG Nb.BbvCI CCTCAGC

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ਊ೧ ༯ᇙ Dam Dcm CpG ^{ਊ೧ ༯ᇙ} Dam Dcm CpG

Nb.BtsI GCAGTG SacI-HF GAGCT/C

NciI CC/SGG ol SacII CCGC/GG

NcoI C/CATGG SalI G/TCGAC

NcoI-HF C/CATGG SalI-HF G/TCGAC

NcoI-HF RE-Mix C/CATGG SalI-HF RE-Mix G/TCGAC

NdeI CA/TATG SapI GCTCTTC(1/4)

NgoMIV G/CCGGC Sau3AI /GATC ol

NheI G/CTAGC scol Sau96I G/GNCC ol ol

NheI-HF G/CTAGC scol SbfI CCTGCA/GG

NheI-HF RE-Mix G/CTAGC scol SbfI-HF CCTGCA/GG

NlaIII CATG/ ScaI-HF AGT/ACT

NlaIV GGN/NCC ol ol ScaI-HF RE-MixAGT/ACT

NmeAIII GCCGAG(21/19) ScrFI CC/NGG ol ol

NotI GC/GGCCGC SexAI A/CCWGGT

NotI-HF GC/GGCCGC SfaNI GCATC(5/9) scol

NotI-HF RE-Mix GC/GGCCGC SfcI C/TRYAG

NruI TCG/CGA ol SfiI GGCCNNNN/NGGCC ol scol

NsiI ATGCA/T SfoI GGC/GCC scol

NspI RCATG/Y SgrAI CR/CCGGYG

Nt.AlwI GGATC(4/-5) SmaI CCC/GGG

Nt.BbvCI CCTCAGC(-5/-7) scol SmlI C/TYRAG

Nt.BsmAI GTCTC(1/-5) SnaBI TAC/GTA

Nt.BspQI GCTCTTC(1/-7) SpeI A/CTAGT

Nt.BstNBI GAGTC(4/-5) SpeI RE-Mix A/CTAGT

Nt.CviPII (0/-1)CCD SpeI-HF A/CTAGT

PacI TTAAT/TAA SphI GCATG/C

PacI RE-Mix TTAAT/TAA SphI-HF GCATG/C

PaeR7I C/TCGAG SspI AAT/ATT

PciI A/CATGT SspI-HF AAT/ATT

PflFI GACN/NNGTC StuI AGG/CCT ol

PflMI CCANNNN/NTGG ol StyI C/CWWGG

PhoI GG/CC scol scol StyI-HF C/CWWGG

PleI GAGTC(4/5) scol StyD4I /CCNGG ol ol

PluTI GGCGC/C SwaI ATTT/AAAT

PmeI GTTT/AAAC scol TaqI T/CGA ol

PmlI CAC/GTG TfiI G/AWTC scol

PpuMI RG/GWCCY ol TseI G/CWGC scol

PshAI GACNN/NNGTC scol Tsp45I /GTSAC

PsiI TTA/TAA TspMI C/CCGGG

PspGI /CCWGG TspRI NNCASTGNN/

PspOMI G/GGCCC scol ol Tth111I GACN/NNGTC

PspXI VC/TCGAGB XbaI T/CTAGA ol

PstI CTGCA/G XbaI RE-Mix T/CTAGA ol

PstI-HF CTGCA/G XcmI CCANNNNN/NNNNTGG

PvuI CGAT/CG XhoI C/TCGAG

PvuI-HF CGAT/CG XhoI RE-Mix C/TCGAG

PvuII CAG/CTG XmaI C/CCGGG

PvuII-HF CAG/CTG XmnI GAANN/NNTTC

RsaI GT/AC scol ZraI GAC/GTC

RsrII CG/GWCCG

SacI GAGCT/C

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ʞʇʣʃ˃ʍʵĜʠĪȤᄎȞਗɩȵīć0120-963-650 [email protected] ʹʙ˃ݛࠐି౯

ʇ˅Ĝʣˋʈʄɼʡ

ํळโɄʇ˅Ĝʣˋʈ

ํळโɄʇ˅ĜʣˋʈɁɉĂᄞ੬൏౯ʶĜʃĜɞʯ˅ʺĜʗĜĂʏʈʢ˃༯ᇙɄɃɈDNA^ඣ၀ ɬʱʇʗĜɅ೾໴ȱȹɤĂۆ๙ાཡএɞʗˋʩʇଋཡএɈȹɛɈʂĜʯˋ˂Ĝʟɻˋʈʭ˄Ĝ ʸɬ࿥ݡȳɥăʇ˅Ĝʣˋʈଌঅɬ਄Ƞ௲ਗɅɉʗĜʊʛʠʱʇʗĜɬಜඣȱȹɂȧɈჂ඘༯ ᇙĂʗĜʊʛʠʱʇʗĜෂɁɈɼˋʍĜʠɈ༯৐Ăʭ˄ĜʸɅȾȞɀ෮౲ȳɥྼᅟȦȜɥăڬ ݚɅʇ˅ĜʣˋʈɈಡो࢒ɍʠˁʮ˃ʏʽĜʞɻˋʈɅ࠲ȳɥʪˋʠɬ૲ȳă

ɼˋʍĜʠȤɢɍʱʇʗĜɈ෮౿

ʯˁʑʷʡᄻᅰɈɼˋʍĜʠ

●ʱʇʗĜɅ෸ಞʇ˅ĜʣˋʈɁȧɥDNAඣ၀ɬ੼ɥȹɛĂไಜɄ౫ ঔਊ೧ɬ઴ᅙȱɀʯˁʑʷʡɬಜඣȳɥăɼˋʍĜʠɅ༯৐౯ɬ૩ȹ ȵȹȞ௲ਗɅɉĂᆤ඘Ʌ঱ࠓ౯ɈɄȞ໻ૻ༯ᇙɬᄵȳɥ౫ঔਊ೧ɬೊ

൰ȳɥă౫ঔਊ೧ཱܾɈ੕ไݛȤɢɍʠˁʮ˃ʏʽĜʞɻˋʈɅȾȞ ɀɉ275 - 277ʲĜʐĂɜȱȩɉɾɿʮʍɼʠɬઞ௎ȳɥă

PCR^{દဘᄻᅰɈɼˋʍĜʠ}

●ɼˋʍĜʠɅ༯৐౯ɬ૩ȹȵȹȞ௲ਗɅɉĂไಜɄ౫ঔਊ೧Ɉ໻ૻင ڮɬᄵȳɥʯˁɼʶĜɬಡोȳɥă

●ɕɂɭɃɈ௲ਗĂ໻ૻ༯ᇙɈȞȴɦȥێၫɈჂ඘Ʌ6^{ܶࡊൎݡȢɦɊ}

୥ယɄಜඣৌᆎɬ૲ȳă

●ʇ˅ĜʣˋʈɅ౷ߊ౯ɬྼᅟɂȳɥ௲ਗɉĂQ5^® DNA Polymerase ĪNEB #M0491īɄɃɈʯ˃Ĝʭ˂Ĝʟɻˋʈ࡙༑ɬᄵȳɥʵ˂ʹ

ˁĜʔɬ઴Ƞă

●PCR௸ॣɈ੕ไݛȤɢɍʠˁʮ˃ʏʽĜʞɻˋʈɈʄɼʡˁɼˋɅ ȾȞɀɉ325 - 326ʲĜʐɜȱȩɉɾɿʮʍɼʠɬઞ௎ȳɥă

●PCRદဘɉɺʄ˅Ĝʑʊ˃Ɂ๝࡜܋຦ȱȹDNAʨˋʡɬಜɤ୸ȳȥĂ ʑʬˋʃˁʸɬ઴ᅙȱɀ౼౿ȳɥă

●ไಜɄ౫ঔਊ೧Ɂಜඣȳɥă

ɺʣĜ˃ȱȹʂ˂ʌDNA^{ᄻᅰɈɼˋʍĜʠ}

●ɺʣĜ˃ȱȹʂ˂ʌDNAɬழȯȞඣ၀Īʯ˅ʺĜʗĜɞʵ˂˂ˋʃĜ ɄɃīɈ೾໴Ʌ઴ᅙɁȧɥă

●2^{ȾɈഊၒโɄʂ˂ʌ}DNA^{ɬɺʣĜ˃ȱɀ}5^Ōɘȹɉ3^{Ōໃ୸Ⴢ඘ɬव౰}

ȱȹɜɈɬĂȷɦɅܾȲȹ౫ঔਊ೧ɁಜඣȱȹʱʇʗĜɂˁɼʊĜ ʏʿˋɁȧɥă

●˂ ˋ બ ݛ ȯ ɦ ɀ Ȟ Ʉ Ȟ ʂ ˂ ʌDNAɉĂT4 Polynucleotide Kinase ĪNEB #M0201īɅɢɤ˂ˋબݛɁȧɥă

ํळโɄɺʣĜ˂ˋʈཱܾ

Each Oligo 5 μl (10 μM stock) 10X Ligase Buffer 5 μl

Nuclease-free water to 50 μl

Incubation 85°C for 10 minutes Cool slowly (30 - 60 minutes)

ʱʇʗĜ

●ไಜɄ౫ঔਊ೧ɁಜඣȳɥăʱʇʗĜɈʓ˃ʭˁɼʊĜʏʿˋɬ႕Ȫ ȭɂɈɁȧɥྥ࿥ශჂ඘ɬ੼౰ȳɥਊ೧ɬ઴ᅙȱȹɕȠȦᆯȞă

ං˂ˋબݛ

●ʓ˃ʭˁɼʊĜʏʿˋɬ႕ȪȹɛĂං˂ˋબݛஐᆅȦྼᅟɄ௲ਗȦȜ ɥăNEBɉ2ହᇆɈDNAං˂ˋબݛਊ೧ɬอࢷȱɀȞɥă

ĝ Calf Intestinal PhosphataseĪCIPĂNEB #M0290īɉဍঢ়Ȟ௸ॣݚ Ɂߤ౯ɬ૲ȱĂɕɂɭɃɈNEBufferෂɁ઴ᅙɁȧɥăȹȺȱCIPɉ༂

Ʌɢɥ࿤ߤ౯ݛȦ୸ᅰɄȞȹɛĂˁɼʊĜʏʿˋ೐ɈDNA౼౿Ȧ࿤

ݢज़ɁȜɥăDNAɈ౼౿ৌᆎɬ௫ȬɥȹɛĂైயᆲڬ௫ɈCIPɬ઴ᅙ ȱɄȞɢȠɅȳɥă

ĝ Antarctic PhosphataseĪAPĂNEB #M0289īɉCIPɂວȲ࡙༑ɬ ᄵȳɥȦĂ༂Ʌɢɥ࿤ߤ౯ݛȦݢ༑ɁȜɥăڃܴĪZnīȦྼీɁȜɥă

APɅɢɥං˂ˋબݛཱܾ

Antarctic Phosphatase 1 μl ( 5 units)

DNA 1-5 μg

10X Buffer 5 μl (1 mM final conc.) Nuclease-free water to 20 μl

Incubation

37°C for 15 minutes (5´ extensions/blunt ends) or 60 minutes (3´ extensions) Heat Inactivate

ိߦݛ / ^Ⴢ඘ୌဌ

● ɼˋʍĜʠɜȱȩɉʱʇʗĜɈჂ඘ɈိߦݛɜȱȩɉჂ඘ୌဌȦྼᅟ Ʉ௲ਗȦȜɥă

● ʯ˃Ĝʭ˂Ĝʟɻˋʈ࡙༑ɬᄵȳɥʵ˂ʹˁĜʔɈPCR^{દဘɈ൥င} ယɉိߦჂ඘ɁȜɥă

● T4 DNA PolymeraseĪNEB #M0203īɜȱȩɉKlenowĪNEB #M0210ī ɉĂ5Ōໃ୸Ⴢ඘ɬႰɛਮəĪEcoRI-HFɄɃīĂ3Ōໃ୸Ⴢ඘ɬதࢦȳɥ ĪPstI-HFɄɃīă

● Quick Blunting Kit^ĪNEB #E1201^{īɬ઴ᅙȳɦɊĂ}30^{ယڬ໘Ʌိߦ} ݛȤɢɍ˂ˋબݛȦݢ༑ɁȜɥă

˂ˋબݛ

● ˁɼʊĜʏʿˋɬ਄ȠȹɛɅɉĂவɄȩɂɜێၫɈDNAჂ඘Īɼˋ ʍĜʠɘȹɉʱʇʗĜīɅ5Ō˂ˋબȦശ੭ȳɥྼᅟȦȜɥă

● ʯˁɼʶĜɉฆ௵˂ˋબݛȯɦɀȞɄȞȹɛĂPCR^દဘɉ5^Ō˂ˋબ ɬ࠸ɘɄȞăێၫĂ౫ঔਊ೧ɅɢɥDNAɈಜඣჂ඘ɉ௵Ʌ5Ō˂ˋબ ɬ࠸ɚă

● DNAඣ၀ɉT4 Polynucleotide KinaseĪNEB #M0201īɁ˂ˋબݛ Ɂȧɥă

T4 PNKɅɢɥ˂ˋબݛཱܾ

T4 PNK 1 μl (10 units)

DNA 1-2 μg

10X T4 PNK Buffer 5 μl

10 mM ATP 5 μl (1 mM final conc.) Nuclease-free water to 50 μl

Incubation 37°C for 30 minutes

ɼˋʍĜʠȤɢɍʱʇʗĜɈ౼౿

● ɺʄ˅Ĝʑʊ˃ȥɣɈಜɤ୸ȱɞʑʬˋʃˁʸ౼౿ɬᅙȞɀĂˁɼ ʊĜʏʿˋ೐ɅɼˋʍĜʠȤɢɍʱʇʗĜɬ౼౿ȳɥă

● ฝᅃ๘ɺʄ˅Ĝʑɂ˸-Agarase IĪNEB #M0392īɬဩᅙȱɀDNA

౼౿Ȧݢ༑ɁȜɥă

● ૓ެೀ௎ଗɅɢɥDNAഹ஥ɬ੕ழঔɅᅦȢɥȹɛĂ๝࡜܋຦ষɅɺ ʄ˅Ĝʑʊ˃ɬࠫઍȳɥ੫Ʌɉෳ༜ෳ૓ެೀĪ360 nmīɬᅙȞɥă

signal sequence

gene MCS drug^R promoter

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ʱʇʗĜȤɢɍɼˋʍĜʠɈˁɼʊĜʏʿˋ

●NEBɉ˂ʄĜʔĆʶʑʗĜʷʛʇʑɬྀ༸ȱɀȞɥă௢੧ɉ100ʲĜ ʐɬઞ௎ȳɥă

●ʱʇʗĜćɼˋʍĜʠɈʺ˃྘ɉ1^ć3^ɂȳɥă

●ଆݗɁLigase Buffer^{ɬᅃސȳɥă}

●ိߦჂ඘ɈˁɼʊĜʏʿˋɁɉĂཱܾ૪࠰ɬෳȩȳɥȥ˂ʄĜʔ༏๱

ɬ਒ȩȳɥྼᅟȦȜɥăȜɥȞɉ˂ʄĜʔĆʶʑʗĜʷʛʇʑɬᅙȞɥă

●ˁɼʊĜʏʿˋষɉ࿉௫ɁᇊࢉȱĂवଋ๖ࠓɬ਄Ƞă

●Quick Ligation BufferȜɥȞɉ˂ʄĜʔĆʶʑʗĜʷʛʇʑɬ઴ᅙȳ ɥ௲ਗɉĂवଋ๖ࠓৌᆎȦฝݚȳɥȹɛĂ༂Ʌɢɥ࿤ߤ౯ݛɬȱɄȞă

वଋ๖ࠓ

●ʋˋʬʞˋʠʓ˃Ɉ௢੧ɉ202 - 213^{ʲĜʐɬઞ௎ȳɥă}

●वଋ๖ࠓৌᆎɈʪˋʠɅȾȞɀɉ323^{ʲĜʐɬઞ௎ȳɥă}

●ʀ˄ʇʠ˅ʵ˄ĜʏʿˋɁवଋ๖ࠓɬ਄Ƞ௲ਗĂˁɼʊĜʏʿˋষɈ

౼౿Ȧ࿤ᅟɄElectroLigase^™Ɉ઴ᅙɬైயȳɥă

NEBɈ˂ʄĜʔɅɢɥˁɼʊĜʏʿˋཱܾ

Quick

Ligation Kit T4 DNA Ligase

Instant Sticky-End Master Mix

Blunt/TA Master

Mix

Format kit enzyme master mix master mix

Buffer 2X Quick Ligation Buffer

T4 DNA Ligase Reaction Buffer

5 μl (master mix)

5 μl (master mix) Vector (3 kb) 50 ng 50 ng 50 ng 50 ng

Insert (1 kb) 50 ng 50 ng 50 ng 50 ng

H₂O up to 20 μl up to 20 μl up to 10 μl up to 10 μl

Total volume 20 μl 20 μl 10 μl 10 μl

Incubation 25°C, 5 minutes

16°C, 30 minutes

N/A, instant ligation

25°C, 15 minutes

Gibson Assembly^™Ʌɢɥʇ˅Ĝʣˋʈ

Gibson Assembly Cloning Kitɉʏˋʈ˃ʙʽĜʮཱܾɁPCRඣ၀ɬࠤ ඍɅʱʇʗĜɅ೨əਮɛɥఛȹɄʇ˅ĜʣˋʈၭɁȜɥă౿࿚ɅɉNEB 5-alpha Competent E .coliȦ࿥മȱɀȞɥă௢੧ɉ127ʲĜʐɜȱȩɉ NEBGibson.com^{ɬઞ௎ȳɥă}

ʯˁɼʶĜɈಡो

●PCRඣ၀ɬʱʇʗĜɅৌᆎɢȩȾɄȬɥȹɛɅɉ15 - 25 ntɈࢼฆ༯ ᇙɬݡȢĂTm^ඩȦ48Ŏڬ௫ɅɄɥɢȠɅಡोȳɥă

●Gibson AssemblyɈʯˁɼʶĜɬಡोȳɥȹɛɅɉʂˋˁɼˋʜĜ

˃ɁȜɥNEBuilder^™Ȧ၆ᆀɁȜɥăNEBGibson.comɬઞ௎ȳɥă ʯˁɼʶĜಡोɈʙʽĜʠ˂ɺ˃ɜ৊ާȯɦɀȞɥă

DNAඣ၀ɈPCRഝဍ

●DNAඣ၀ɈഝဍɅɉQ5 High-Fidelity DNA PolymeraseĪNEB #M0491īĂ ɘȹɉʳʛʠʑʗĜʠ઱ᅕĪNEB #M0493īȥʶʑʗĜʷʛʇʑ઱

ᅕĪNEB #M0492īɈ઴ᅙɬైயȳɥă

●ࠞ௻DNAɬʞˋʯ˄Ĝʠɂȳɥ௲ਗĂ50 ̂lɈPCRཱܾȜȹɤ੕ழ ঔɈDNA^Ī0.1 - 0.5 ng^{īɬ઴ᅙȳɥă}

●PCRદဘɈ஋๱ɂେᆲɬʊ˃๝࡜܋຦Ɂߊ໻ȳɥă

●PCR^દဘȦ>90%Ɉ౼౿๱ɁȜɦɊ౼౿ɉྼᅟɄȞă

●౼౿ɬȱɀȞɄȞPCR^દဘɉGibson Assembly^ཱܾɅ20%^ɘɁݡȢ ɥȭɂȦɁȧɥă

●ഽᆲɈʯˁʑʷʡɬʞˋʯ˄ĜʠɅ઴ᅙȳɥ௲ਗĂʨʛʇʈˁɾˋʡ ɬᅦȢɥȹɛɅDpnI^ĪNEB #R0176^īɁPCR^{દဘෂɈʞˋʯ˄Ĝʠ} ʯˁʑʷʡɬಜඣȳɥă

ʱʇʗĜɈೀ௻ݛ

●౫ঔਊ೧ɬᅙȞɀʱʇʗĜɬೀ௻ݛȳɥ௲ਗĂ჎ಜඣʱʇʗĜɈʨʛ ʇʈˁɾˋʡɬฝঌȳɥȹɛɅ2ହᇆɈ౫ঔਊ೧ɬᅙȞɀʘʮ˃ʘɼ ʐɿʑʠɬ਄Ƞȭɂɬైயȳɥă

●ʱʇʗĜDNA^ɈഝဍɅɉQ5 High-Fidelity DNA Polymerase^ĪNEB

#M0491īĂɘȹɉʳʛʠʑʗĜʠ઱ᅕĪNEB #M0493īȥʶʑʗĜ ʷʛʇʑ઱ᅕĪNEB #M0492^{īɈ઴ᅙɬైயȳɥă}

ཱܾʓʛʠɺʛʯȤɢɍཱܾ௸ॣ

●౼౿ȱȹDNAɬ౉ĪMilli-Q౉ȥວຑɈɜɈīĂTEȜɥȞɉ഼Ɉʨʛ ʭɹĜɅᅗȥȳă

● ੕ไɄ௸ॣɁɺʛʓˋʮ˃ɬ਄ȠȹɛɅĂGibson Assembly Master Mix^{ɬݡȢɥ೐Ʌ}DNA^ᅗܕɬ10 ^̂lɂɄɥɢȠɅ෮౲ȳɥă

● 1^ɜȱȩɉ2^ହᇆɈDNAඣ၀ɬʱʇʗĜɅȾɄȬɥ௲ਗĂʠĜʗ˃Ɂ 0.02 - 0.5 pmolɈDNAඣ၀ɬ઴ᅙȳɥȭɂɬైயȳɥă

Gibson Assembly^{ʇ˅ĜʣˋʈၭɈޱᆒ}

● DNAᅗܕɈᅎᆲȦ10 ̂lڬ௫ɁȜɥ௲ਗĂGibson Assembly Master MixɈᅎᆲɬไࡱ෮౲ȳɥă

● Gibson Assembly Cloning Kitɉ50ŎĂ15ယ࠰Ɂɺʛʓˋʮ˃ȳɥ ɢȠɅ੕ไݛȯɦɀȞɥă15ယڬ௫ɈɼˋʅʽʱĜʏʿˋɉྼᅟɄ ȞȦĂ௲ਗɅɢȽɀɉɼˋʅʽʱĜʏʿˋ૪࠰ɬෳȩȳɥȭɂɁĪ60 ယ࠰īɺʛʓˋʮ˃ৌᆎȦ৐௫ȳɥȭɂȦ૲ȯɦɀȞɥă

● ɺʛʓˋʮ˃ཱܾɬʂĜʨĜʢɼʠɁ਄ɩɄȞă

NEB 5-alpha Competent E. coliĪHigh EfficiencyīɒɈवଋ๖ࠓ

● ɺʛʓˋʮ˃ȱȹDNAȦ20 kb჎჊ɁȜɥ௲ਗĂʅʛʠɅ࿥മȱɀȞ ɥNEB 5-alpha Competent E. coliĪHigh Efficiencyīɬ઴ᅙȳɥȭ ɂɬైயȳɥă

● ࢍၫ৐ཱဌ༯ᇙĪInverted repeatīɞʗˋʟʸཱဌ༯ᇙĪtandem repeatīɬ࠸ɚȜɥହɈ৭ഢɬᄵȳɥDNAɉ൥෬ࣳ໘Ɂೊ်ȯɦĂ वଋ๖ࠓৌᆎɈฝݚɞʋ˅ʣĜɈழळݛɬۜȧࡧȭȳȭɂȦȜɥă

● ʀ˄ʇʠ˅ʵ˄Ĝʏʿˋɬ઴ȢɊवଋ๖ࠓৌᆎɬౘख़৐௫Ɂȧɥăʀ

˄ʇʠ˅ʵ˄Ĝʏʿˋɬ਄Ƞ௲ਗĂGibson Assemblyཱܾᅗܕɬ3༰ ɅࡏଫȱɀĂ1 ̂lɬवଋ๖ࠓɅ઴ᅙȳɥă

+

Analyze plasmid DNA from 4-8 individual colonies by colony PCR or restriction enzyme digestion,

and/or sequence the insert.

1. Generate a linear vector.

Linear vector

Linear vector

฀ ฀ ฀ ฀ ฀ ฀

฀ ฀ ฀

฀ ฀ ฀ ฀ ฀

฀ ฀ ฀ ฀

2. PCR amplify DNA inserts with overlapping ends.

3. Add fragments and linear vector to Gibson Assembly Master Mix, and incubate at 50°C for 15 minutes.

฀

4. Transform NEB 5-alpha CompetentE. coli with 2 µl Gibson Assembly reaction.

฀ sequences

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ʞʇʣʃ˃ʍʵĜʠĪȤᄎȞਗɩȵīć0120-963-650 [email protected] ʹʙ˃ݛࠐି౯

ʇ˅ĜʣˋʈɈʠˁʮ˃ʏʽĜʞɻˋʈʄɼʡ

ݚࡥ࿍ɅɉĂယાʇ˅ĜʣˋʈଌঅɅȤȫɥʠˁʮ˃ʏʽĜʞɻˋʈȦ

૲ȯɦɀȞɥăवଋ๖ࠓཱܾɈ੕ไݛɈʪˋʠɅ࠲ȱɀɉ323^ʲĜʐɬ ઞ௎ă

ᄎ൨๘ ঈۚ ޜ೑ၫၭ

वଋ๖ࠓൌȦ ɕɂɭɃຶɣɦɄȞĂ ɘȹɉ

ɘȽȹȩຶɣɦɄȞ

ˁɼʊĜʏʿˋȦ࿤୥ယ

● ಜඣȱɀȞɄȞʱʇʗĜȤɢɍێݳஒಜඣȱȹʱʇʗĜɅĂT4 DNA Ligaseɬ๒ݡȱɀĂ ɘȹɉ๒ݡȵȴɅɼˋʅʽʱĜʏʿˋȱȹʋˋʠ˅Ĝ˃ཱܾृɬᅙȞɀߊ໻ȳɥăɘȹɉ

́DNA-HindIII DigestĪNEB #N3012īɞ഼ɈไಜɄࡊଋɬᅙȞɀT4 DNA Ligaseߤ౯ ɬ૚অȳɥă

● ʱʇʗĜćɼˋʍĜʠɈʺ˃྘ɬ1^ć1^ȥɣ1^ć15Ɉ࠰Ɂဿ৩ȱɀ੕ไݛȳɥă

● ʨʛʭɹĜෂɈATPɞDTTȦယސȯɦɀȞɥȭɂȦȜɥȹɛĂఛȱȞʨʛʭɹĜɬᅙȞɥă

● DNAɬ౼౿ȱɀˁɼʊĜʏʿˋཱܾɅࢷȳɥĪNaClɞEDTAɄɃɈᐍઔဘȦৌᆎɬฝݚ ȯȵɥīă

● ʋˋʃʞʶĜɈव౰ɬྤȫɥȹɛĂ೔ཱܾܕෂɈDNA༏๱ɬ10 ng /̂lڬݚɅȳɥă

● 1ܶࡊໃ୸Ⴢ඘ɈˁɼʊĜʏʿˋɅɉĂBlunt / TA Ligase Master MixĪNEB #M0367īȥĂ Quick Ligation KitĪNEB #M2200īĂ਒༏๱LigaseĪNEB #M0202TīȦྼᅟɄ௲ਗȦ Ȝɥă

● PEGĪQuick Ligation^™ KitɈʨʛʭɹĜɅ࠸ɘɦɀȞɥīɉʂĜʨĜʢɼʠ૪ɈˁɼʊĜ ʏʿˋཱܾৌᆎɈฝݚɂʀ˄ʇʠ˅ʵ˄ĜʏʿˋৌᆎɈฝݚɬۜȧࡧȭȳȭɂȦȜɥȹ ɛĂʀ˄ʇʠ˅ʵ˄Ĝʏʿˋ೐ɅʑʬˋʃˁʸɁPEGɬதࢦȳɥă

● ʱʇʗĜɈಜඣჂ඘Ɉഹ஥ɬߊ໻ȳɥăߊ໻ɉێݳஒಜඣȱȹʱʇʗĜɬං˂ˋબݛȱĂ T4 Polynucleotide KinaseĪNEB #M0201īɂɼˋʅʽʱĜʏʿˋȱȹɜɈɬˁɼʊĜ ʏʿˋȳɥă

● ˂ʄĜʔɬ༂Ʌɢɤ࿤ߤ౯ݛȳɥȭɂɁवଋ๖ࠓৌᆎȦ৐௫ȳɥݢ༑౯ȦȜɥăȹȺȱ Quick Ligation KitȤɢɍLigase Master MixɉʨʛʭɹĜෂɅPEGɬ࠸ɚȹɛĂ༂Ʌɢɥ

࿤ߤ౯ݛȦɁȧɄȞă

ಜඣȦ࿤ࠊ೔

● ɺʄ˅Ĝʑʊ˃๝࡜܋຦ɅɢɤʱʇʗĜɈಜඣɬߊ໻ȳɥăʱʇʗĜɈಜඣȦ࿤୥ယɁ ȜȽȹ௲ਗĂ੔๱ಜඣɬ਄ȠȥĂಜඣȯɦȹʱʇʗĜɬಜɤ୸ȱɀ઴ᅙȳɥă

● வɄȩɂɜ6খɈʤʇ˄ʂʙʡɬ౫ঔਊ೧ငڮɈ௫ᆔɅ࿥ݡȱȹʯˁɼʶĜɬ੔ಡोȳɥă ಜඣჂ඘ȦഊၒโɁɄȞ ^● ʱʇʗĜĂɼˋʍĜʠȤɢɍʯˁɼʶĜɈ༯ᇙෂɅᄆโɂȳɥ౫ঔငڮȦȜɥȭɂɬߊ໻

ȳɥă

˂ˋબࡊɈज़໵

● ˁɼʊĜʏʿˋɅɉʱʇʗĜȜɥȞɉɼˋʍĜʠɈ5Ō˂ˋબȦྼᅟɁȜɥă˂ˋબࡊȦശ ੭ȱɄȞ௲ਗɉĂT4 Polynucleotide KinaseĪNEB #M0201īɁʱʇʗĜɘȹɉɼˋʍĜ ʠɬ˂ˋબݛȳɥă

౼౿ݿาɁ DNAȦഹ஥ȱɀȞɥ

● ૓ެೀɒɈႉᇮɬ੕ழঔɅᅦȢɥȹɛĂෳ༜ෳ૓ެೀĪ360 nmīɬᅙȞɀɺʄ˅Ĝʑʊ

˃ɬࠫઍȳɥăɘȹฝᅃ๘ɺʄ˅Ĝʑɂ˸-Agarase IĪNEB #M0392īɅɢɥஐᆅষɅĂ DNA෺ਏɞʑʬˋʃˁʸஐᆅɬ਄Ƞă

DNAჂ඘Ȧ

౷ȱȩိߦݛȯɦɀȞɄȞ

● T4 DNA polymeraseĪNEB #M0203īɞKlenowĪNEB #M0210īĂQuick Blunting Kit ĪNEB #E1201īɬᅙȞɀĂɼˋʍĜʠɞʱʇʗĜɈჂ඘ɬိߦݛȳɥă

ɼˋʍĜʠɅɢɥ ੧ၲຽ౯Ȧࡷɩɦɥ

● ཡ এ ɬ উ დ Ʌ ౫ স Ɂ ȧ ɥ ʋ ˋ ʬ ʞ ˋ ʠ ʓ ˃ ࣳ ߰ ɬ ઴ ᅙ ȳ ɥĪNEB 5-alpha FŌI^q Competent E. coli, NEB #C2992ɄɃīăɘȹɉɼˋʅʽʱĜʏʿˋݗ๱ɬ30ŎɘɁݚȬɥă वଋ๖ࠓৌᆎȦ੕ไɁɉɄȞ ^● वଋ๖ࠓཱܾɈ੕ไݛȤɢɍʠˁʮ˃ʏʽĜʞɻˋʈɈʪˋʠɅȾȞɀ323ʲĜʐɬઞ௎

ȳɥă वଋ๖ࠓൌɅऋʱʇʗĜ

ȱȥ࠸ɘɦɀȞɄȞ ĪɼˋʍĜʠȦ

೾໴ȯɦɀȞɄȞī

ʱʇʗĜɈ ʓ˃ʭˁɼʊĜʏʿˋ

● Calf Intestinal PhosphataseĪNEB #M0290īɘȹɉAntarctic PhosphataseĪNEB #M0289ī ɅɢɤĂʱʇʗĜDNAɬං˂ˋબݛȳɥă

ʱʇʗĜɈಜඣȦ࿤୥ယ ^● ˁɼʊĜʏʿˋɬ਄ɩȴɅʱʇʗĜඣ၀ɬवଋ๖ࠓȱɀߊ໻ȳɥăಜඣȦ࿤୥ယɄ௲ਗĂ

੔๱ಜඣɬ਄ȠȥĂ෸੍௻ʱʇʗĜɬ౼౿ȳɥă

वଋ๖ࠓൌɅ࠰ۅȽȹ ʯˁʑʷʡȦ

࠸ɘɦɀȞɥ

࠰ۅȽȹPCRદဘɬ

઴ᅙȱɀȞɥ ^● PCRદဘɬʊ˃౼౿ȳɥă

ྥູڽโɄPCRદဘȦ

਼໴ȱɀȞɥ ^● PCRદဘɬʊ˃౼౿ȳɥă

ʏĜʉˋʑʀˁĜ ^● ਒౷ߊ౯ʵ˂ʹˁĜʔɬ઴ᅙȱɀPCRඣ၀ɬഝဍȳɥĪQ5 High-Fidelity DNA PolymeraseĂ NEB #M0491ɄɃīă

౷ȱȩɄȞʯˁɼʶĜɬ

઴ᅙȱɀȞɥ ^● ʯˁɼʶĜ༯ᇙɬߊ໻ȳɥă ɼˋʍĜʠɅɢɥ

੧ၲຽ౯Ȧࡷɩɦɥ

● ཡ এ ɬ উ დ Ʌ ౫ স Ɂ ȧ ɥ ʋ ˋ ʬ ʞ ˋ ʠ ʓ ˃ ࣳ ߰ ɬ ઴ ᅙ ȳ ɥĪNEB 5-alpha FŌI^q Competent E. coli, NEB #C2992īăɘȹɉɼˋʅʽʱĜʏʿˋݗ๱ɬ30ŎɘɁݚȬɥă वଋ๖ࠓൌɅ

ʯˁʑʷʡȦ

࠸ɘɦɀȞɄȞ

ৣ౺ဘଋ༏๱ȦฝȳȨɥ ^● ༱ණෂɈৣ౺ဘଋ༏๱ɬ௫Ȭɥă

ʍʞˁɼʠʋ˅ʣĜɬ઴ᅙȱȹ ^● ɢɤ൥ȧȩĂ౺ۋ஑ࡖɅ୸এȱȹʋ˅ʣĜɬೊ်ȳɥă

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ۆ๙ાʶĜʃĜ

ۆ๙ાळĪgenotypeīɂɉĂȜɥ౺ဘখൌɅȤȫɥۆ๙ߔโɄDNAɈ௻ൔɈȭɂɁȜɥăۆ๙ા

ळɉȜɥ߰ɁࠫઍȯɦɥवଋĪ࿍এळĂphenotypeĂݚࡥઞ௎īɬड़ฤȳɥۆ๙ાߔโ௻ൔɬ࿍

ȳᆅሄโɄ࿍ࡥၭɁȜɥă൥෬ࣳɈۆ๙ાळɁɉ࡙༑Ȧज़ഹȱȹۆ๙ાɈəȦࡥ੪ȯɦɥĪ1īă ࡥ੪ȯɦɀȞɄȞۆ๙ાɅȾȞɀɉĂဿڽ*^ƵȱɀȞɥȥɃȠȥ࿤ცɁȜɥăʂ˂ʐʢ˃ɈK-12^߰ ෂɅശ੭ȱɀȞȹʯ˅ʭɹĜʐȤɢɍʯˁʑʷʡĪFẮĂe14ĂracīɉĂज़ഹȱɀȞɥ௲ਗɅɈ əࡥ੪ȯɦɥɈȦێཾโɁȜɥăȭȭɁɉ၆ࡱ௫ẮɅȾȞɀɉശ੭ȳɥ௲ਗɅɈəࡥ੪ȱĪശ ੭ȱɄȞɂȧɅɉࡥ੪ȱɄȞīĂFȤɢɍȷɈဿڽळɅȾȞɀɉȳɓɀࡥ੪ȱɀȞɥăʯ˅ʭɹĜ ʐɞʯˁʑʷʡɉĪīɘȹɉĮįɁڱɭɁࡥ੪ȱɀȞɥăۆ๙ાɉĂȷɈ࡙༑ɬૃȞࡧȭȱɞȳ Ȟழဦ૦3ဦ૦ɬᅙȞɀɼʗ˂ʛʇൌɁ࿍ࡥȯɦɥĪᇉĂdamćDNA adenine methylaseīăထౘ Ɉۆ๙ાȦວȲ࡙༑Ʌ࠲ᅇȳɥ௲ਗɅɉĂɼʗ˂ʛʇൌɈ൥ဦ૦ɬฃݡȱɀڽɄɥۆ๙ાɬ်ࣾ

ȳɥĪᇉćrecAĂrecBĂrecCĂrecDĂȭɦɣɉȳɓɀrecombinationĮۆ๙ા೨ࠓȢįɅ࠲ᅇȳ ɥīăۆ๙ાळɈ࿍ࡥɁɉ௫࿥ȧɈ+^ɞ–ɉௐᆒȯɦɥɈȦێཾโɁȜɥȦĂۆ๙ાळɬცߊɅȳɥ ȹɛɅ୭ထȱɀ઴ᅙȯɦɥȭɂȦȜɥĪᇉćFŌlac-proA⁺B⁺īăज़ഹဿڽൌɉΔɁ࿍ࡥȱĂȷɦɅറ ȞɀĪī໘Ʌज़ഹۆ๙ાშɬ࿍ࡥȳɥĮᇉćΔĪlac-pro^īįăშ೐Ȧ࿍ࡥȯɦȹۆ๙ાɈ࠰Ʌശ੭ ȳɥۆ๙ાɉĂȳɓɀज़ഹȱɀȞɥăᇉȢɊĂΔĪlac-proīɈ௲ਗɉlacɂproɈ࠰Ɉۆ๙ાȦȳɓ ɀज़ഹȱɀȞɥȭɂɬ૲ȳăູฤɈဿڽɉĂɼʗ˂ʛʇൌɈɺˁʫɺౘ૦Ʌɢɤൎᆏۆ๙ાྈਖ ĪᇉćhsdR17īɁ࿍ࡥȯɦĂྼᅟɅܾȲɀam = ɺˋʨĜĪUAGīဿڽɘȹɉts = ݗ๱ࠐି౯Ī਒

ݗɁ࿤ߤ౯īဿڽɈɢȠɅ࿍ࡥȯɦɥăێཾโɄൎᆏۆ๙ાɉĂȭɈ࿍ࡥ˃Ĝ˃Ʌ୦ɩɄȞɜɈ ɜȜɥĮᇉćΔĪlac-proīX111įă2ହᇆɈ߰Ɉۆ๙ાळɅວȲൎᆏۆ๙ાྈਖȦ࿍ࡥȯɦɀȞɥ

௲ਗĂȷɦɣɉ೔ȩວȲဿڽɬᄵȳɥɉȴɁȜɥă

࿍এळĪphenotypeīɂɉĂࠫઍݢ༑Ʉࢫ຦ĪवଋīɈȭɂɁȜɥăᇉȢɊĂLac^{–ɉඖ೧঍Ȧˁ}

ʇʠĜʑɈəɁɉ౺ۋȳɥȭɂȦɁȧɄȞă࿍এळɉ൥ဦ૦Ɂ઻ɘɥ˅ĜʶൌɁ࿍ࡥȯɦĂဦ૦ Ʌɉ௵Ʌ௫࿥ȧɈ+^ɘȹɉ–Ȧ࿥ȩĪ൏౯ɘȹɉࠐି౯ɬڷჍȳɥr^ɘȹɉs^{Ȧ࿥ȩ௲ਗɜȜɥīăউ} დɅ঒ȢɊĂ࿍এळɉۆ๙ાळɂێ஗Ʌࡥ੪ȳɓȧɁɉɄȞȦĂۆ๙ાळȥɣ࿍এळɬცߊɅᅅ೻ȳ ɥȭɂȦɁȧɄȞ௲ਗɅɉĂۆ๙ાळɈষɅ࿍এळɬࡥ੪ȳɥȭɂȦȜɥăĮᇉćrpsL104^ĪStr^rī Ɉۆ๙ાშɉ˂ʴʕĜʸʗˋʩʇଋழʍʮʾʣʛʠĪribosomal protein, small subunitīɈS12 ɅᄻᅰȱĂʑʠ˄ʯʠʶɼʏˋ൏౯ɬᅇȢɥįă

ڬݚȤɢɍ૫ʲĜʐɅێཾโɄۆ๙ાळɬࡥ੪ȳɥăۆ๙ߔโɅฤࡹȯɦɀȞɥۆ๙ાɉઞ৽ဦ ॷ2Ʌࡥ੪ȯɦɀȞɥ഼ĂʀĜ˃൥ߔɈE. coli Genetic Stock CenterĪCGSCīȦࠣᆅȳɥྥ௵

ɅᄵᅙɄɼˋʗĜʥʛʠʍɼʠIJhttp://cgsc.biology.yale.edu/ijɁɜઞ௎ȳɥȭɂȦɁȧɥă CGSCɈ௢੧ɅȾȞɀɉĂಗ໸ଞɁȜɥMary Berlyn્IJ[email protected]ijɅᄎȞਗɩȵɣɦȹȞă

* ॸ࢞ଆɁ઴ᅙȯɦɀȞɥɕɂɭɃɈ൥෬ࣳ߰ɉݿࢦ40༃ڬ௫Ʌɩȹɥॸ࢞ɁഽౘɈဿڽȦຩ໴ȯɦɀȧȹăȷɦ ȸɦɈ߰ɅɉȭɦɘɁɅཡॽȯɦɀȞɄȞဿڽȦശ੭ȳɥݢ༑౯ȦȜɥăȷɈɢȠɄဿڽȦଌঅय़ݪɅ܆ࣗɬ࢒

ɖȳȥɃȠȥɉ࿤ცɁȜɥăȱȹȦȽɀĂଌঅɅɉ1ହᇆڬ௫Ɉ߰ĂɘȹɉʨʛʇʈˁɾˋʡɈڽɄɥ߰ɬ઴ᅙ ȳɥȭɂȦৌݪโɁȜɥă

Ƶ൥෬ࣳB߰ȤɢɍȷɈ༝౺߰ɉĂ౺ᅰLon^–ȤɢɍDcm^–ɁȜɥăȭɦɣɈ߰ɁɉȭɈ࿍এळȦᄘ౺ळɁȜɥȦĂ ȭȭɁɉȜȢɀĪ ī໘Ʌ࿍ࡥȱɀȞɥă

ઞ৽ဦॷ

(1) Demerec et al. (1966) Genetics, 54, 61-76.

(2) Berlyn, M.K.B. (1996). In F. C. Niedhardt et al. (Ed.), Escherichia coli and Salmonella:

cellular and molecular biology, (2nd ed.), Vol. 2, (pp. 1715-1902). ASM Press.

(3) Raleigh, E.A. et al. (1991) J. Bacteriol., 173, 2707-2709.

dam GATC༯ᇙɬ໻ૻȳɥ໘ۚ౯ɺʟʣˋʹ

ʙ˃ݛߤ౯Ȧज़ഹȱɀȞɥădam߰ɉ਒

Ȟ࿡๱Ɂۆ๙ા೨əࠓȢɬࡧȭȱĂৡ௵

โ ɅDNAୌ ౷ ߤ ౯ ɬ ཡ এ ȳ ɥă ɘ ȹĂ DamୌఄʯˁʑʷʡɅɢɥवଋ๖ࠓȦȯ ɦɅȩȞăDamʹʙ˃ݛࠐି౯Ɉ౫ঔਊ ೧ĪᇉćBclIīɬᅙȞɀDNAɬಜඣȳɥ

௲ਗɅ઴ᅙȳɥă

dcm CCWGG༯ᇙɬ໻ૻȳɥ໘ۚાʏʠʏˋ

ʹʙ˃ݛߤ౯Ȧज़ഹȱɀȞɥăDcmʹʙ

˃ݛࠐି౯Ɉ౫ঔਊ೧ĪᇉćAvaIIīɬᅙ ȞɀDNAɬಜඣȳɥ௲ਗɅ઴ᅙȳɥă dnaJ ȞȩȾȥȜɥĶʏʻʲ˅ʣˋķɈ1ȾȦ

࿤ߤ౯ݛȱɀȞɥăȭɈज़ഹɅɢɤĂ൥

෬ࣳɁཡএȯɦɥဿڽʗˋʩʇଋȦڤฤ ݛȯɦɥȭɂȦ૲ȯɦɀȞɥă dut dUTPaseߤ౯Ȧज़ഹȱɀȞɥăungɂ೨

əਗɩȵɥȭɂɅɢɤĂɾˁʏ˃ɬDNA ɅଲɤਮɘȵɥȭɂȦɁȧɥăʂ˂ʌʤ ʇ˄ʂʙʡဿڽຩ໴Ɉഀ੼Ɂ઴ᅙȯɦɥ ȭɂȦȜɥă

endA ྥູڽโʀˋʡʤʇ˄ɺĜʔ I ߤ౯Ȧज़ ഹ ȱ ɀ Ȟ ɥăendA߰ ɢ ɤ ෮ ౿ ȯ ɦ ȹ DNAɉ਒࿚ଋɁȜɥɂ৽ȢɣɦɀȞɥă e14 ಜɤ୸ȱݢ༑Ʉʯ˅ʭɹĜʐᅕۚાɁȜ ɤĂK-12߰Ʌɉശ੭ȳɥȦഽȩɈ༝౺߰

Ɂɉज़ᅶȱɀȞɥăe14ɉmcrAɄɃɈۆ ๙ાɬ။ᄵȳɥȹɛĂe14^– ߰Ɉ࿍এळɉ McrA^– ɂɄɥă

F ฝʋʬĜౘɈ૵জ๙༙౯ʯˁʑʷʡɁ ȜɥăFŌۚાɉ൥෬ࣳಹఋൌɈێငယɬ

။ᄵȱĂູ྾ȯɦɥۆ๙ાɂȱɀɉFŌ lacproA⁺B⁺ ௫Ɉlacʂʲ˅ˋȤɢɍproAB ȦȜɥă

fhuA ์ʠˁˋʑʵĜʗĜିᅎൌĪferric hydrox-amate uptakeīɈဿڽൌɬ။ᄵȱĂT1 ʭɹĜʐࠐಹɅൎȱɀ൏౯ɬ૲ȳăȭɦ ɘɁɉtonAɂȱɀඪɣɦɀȞȹă gal ʄˁʇʠĜʑɈൣଟ༑ɬज़ഹȱɀȞɥă glnV supE^ɬઞ௎ă

gyrA DNAʐʻɼ˄ĜʑɈǝʍʮʾʣʛʠɅ๘

ဿڽɬ။ᄵȳɥăȭɈဿڽɉৣ౺ဘଋɁ Ȝɥʢ˂ʐʇʑબɅൎȳɥ൏౯ɬɜȹ ɣȳă

hflA ́ɅɢɥᅗঈݛĪ൥෬ࣳಹఋൌɒɈଲɤ ਮəīɈ࿡๱Ȧ਒ȩɄɥă

hsdR, ູฤɈ༯ᇙȦʹʙ˃ݛȯɦɀȞɄȞDNA

ɉĂEcoKIɘ ȹ ɉEcoBIɅ ɢ Ƚ ɀ ެ ᅰ DNAɂ໻ૻȯɦĂ౫ঔĪಜඣīȯɦɥă ȭ ɦ ɣ Ɉ ਊ ೧ ɉ ڽ Ʉ ɥ ༯ ᇙ ɬ ໻ ૻ ȱĂ hsdRMSۆ๙ા௫ɈڽɄɥൎᆏۆ๙ાɅ ɢɤʋĜʡȯɦɀȞɥăhsdRဿڽɉ౫ঔ ယސɬഹɄȠȦĂ။ীโʹʙ˃ݛɉഹɄ ɩɄȞĪr^– m⁺īăێၫĂhsdSဿڽൌɉᆤ

ၫɬഹɄȠĪr^– m^– īăhsdS߰Ɂ੼ɣɦȹ DNAȦᄘ౺߰Ʌवଋ๖ࠓȯɦɥɂĂ౫ঔ Īಜඣīȯɦɥă

hsdS

321

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12

ʞʇʣʃ˃ʍʵĜʠĪȤᄎȞਗɩȵīć0120-963-650 [email protected] ۆ๙ાʶĜʃĜ

ۆ๙ાʶĜʃĜĪറȧī

mcrBC

lacI^q lac˂ʯ˄ʛʍĜɬݿ௰દ౺ȱĂP lacĪlac ʯ˅ʺĜʗĜīȥɣɈཡএɬɢɤࠊ೔Ʌ ᅦ౫ȳɥă

lacZ ˸-ʄˁʇʠʏʘĜʔߤ౯Ȧज़ഹȱɀȞɥă lacZ:: lacZۆ๙ાɅʭɹĜʐᄻᅰɈT7 RNAʵ

˂ʹˁĜʔۆ๙ાĪ= gene1īȦ೾໴ȯ ɦɀȞɥă

lacY ˁʇʠĜʑʩĜʷɺĜʔߤ౯Ȧज़ഹȱɀ Ȟɥă

ΔĪlacī= ज़ഹĈlacɅ࠲ᇦȳɥێཾโɄ ज़ഹȦǐݳஒȜɥć

ΔĪlacZīM15ɉĂᅕęɄʱʇʗĜɅʋĜ ʡȯɦɀȞɥlac˷-ʭˁʈʹˋʠɬၒ๎

ȳɥʭˁʈʹˋʠɬཡএȳɥăɢȽɀĂ ȭɦɣɈʱʇʗĜɉୱ଱ȦΔM15ɬ။ᄵ ȳɥ௲ਗɅɈəX-Gal௫Ɂಆఋɬ౺Ȳɥă ΔU169ĂΔX111ĂΔX74ɉȞȴɦɜlacʂ ʲ˅ˋȦಹఋൌȥɣࠊ೔Ʌज़ഹȱɀȤ ɤĂȷɈୈ၃ɈDNAɜᅕęɄෳȯɁज़ഹ ȱɀȞɥăΔX111ɉproABɜວ૪Ʌज़ഹ ȱɀȞɥɈɁĂFŌlacproA⁺B⁺ ɬ။ᄵȱɄ ȞঔɤĂ൥෬ࣳ߰Ȧ੕வ༱ණɁ౰ෳȳɥ ȹɛɅʯ˅˂ˋɬᅟ࢙ȳɥă lon ڽ௵ʗˋʩʇଋɬယސȳɥʯ˅ʞɺĜʔ

ߤ౯ɬज़ഹȱɀȞɥăȜɥହɈణ߇౺ဘ ʗˋʩʇଋɉlon߰໘Ɂڤฤݛȳɥă൥෬

ࣳB߰ɉ౺ᅰLonɬज़ഹȱɀȞɥă

lysY T7ʨʇʞ˂ʂʭɹĜʐᄻᅰɈဿڽळ˂ʖ

ʙĜʸɬʋĜʡȱɀȞɥăK128Yဿڽɉ

˂ʖʙĜʸߤ౯ɬज़଄ȯȵɥȦĂဿڽळ ʗˋʩʇଋɉT7 RNAʵ˂ʹˁĜʔɅय़ ਗȱ೫ޮȳɥă

malB malBᆴ ۊ Ʌ ɉĂ ۆ ๙ ાmalEFGȤ ɢ ɍ malK lamB malMȦശ੭ȳɥăΔĪmalBī ɉȭɈᆴۊɈႤɃȳɓɀɬज़ഹȱɀȤ ɤĂʶ˃ʠĜʑय़ਗʗˋʩʇଋĪMalEī ȦཡএȱɄȞă

mcrA, ʹʙ˃ʏʠʏˋູڽโɄ౫ঔʏʑʞʸȦ

ज़ഹȱɀȞɥăȜɥ༯ᇙɅʹʙ˃ʏʠʏ ˋɬᄵȳɥDNAɉĂMcr⁺ ɅɢȽɀಜඣ ȯɦɥădcm^– ୌఄȯɦȹDNAĪʏʠʏ ˋ Ȧ ʹ ʙ ˃ ݛ ȯ ɦ ɀ Ȟ Ʉ ȞDNAī ɉ Mcr⁺ ɅɢȽɀಜඣȯɦɄȞăΔĪmcrC- mrrīɉmcrC-mcrB-hsdS-hsdM-hsdR-mrrɈ6Ⱦ Ɉ ۆ ๙ ા ɬ ज़ ഹ ȱ ɀ Ȟ ɥă mcrAɉe14Ɉज़ഹɅ཮Ȟज़ഹȳɥă mrr ʹʙ˃ʏʠʏˋȜɥȞɉʹʙ˃ɺʟʣˋ

ູڽโɄ౫ঔʏʑʞʸȦज़ഹȱɀȞɥă ȹȺȱĂdam^–Ădcm^– ȜɥȞɉEcoKIୌ

ఄȯɦȹDNAĪʏʠʏˋȜɥȞɉɺʟʣ ˋȦʹʙ˃ݛȯɦɀȞɄȞDNAīɉMrr⁺ ɅɢȽɀಜඣȯɦɄȞăʹʙ˃ʏʠʏˋ گശ౯Ɉߤ౯ɉMcrFɂȱɀɜඪɣɦɀȞ ɥĪ3īă

mtl ດɺ˃ʋĜ˃ɁȜɥʶˋʣʠĜ˃ൣଟ༑

Ȧज़ഹȱɀȞɥă

ompT ެႸʯ˅ʞɺĜʔĪʯ˅ʞɺĜʔVIIīߤ ౯Ȧज़ഹȱɀȞɥă

phoA ɺ˃ʃ˂ʳʑʭɹʗĜʔߤ౯Ȧज़ഹȱɀ Ȟɥă

prc tsp^ɬઞ௎ă

recA ഊວ೨ࠓȢߤ౯Ȧज़ഹȱɀȞɥă50 bp ɬ෰Ȣɥ෸ᇙཱဌɬ࠸ɚ༯ᇙɬʇ˅Ĝʣ ˋʈȳɥ੫ɅɉĂȭɈۆ๙ાळɬɜȾ߰

Ȧ৕ɘȱȞă

recB, ʀ ʅ ʕ ʤ ʇ ˄ ɺ Ĝ ʔVɈ ʀ ʅ ʕ ʤ ʇ ˄ ɺĜʔߤ౯Ȥɢɍ೨ࠓȢߤ౯Ȧज़ഹȱɀ Ȟ ɥăsbcBɘ ȹ ɉsbcAɁ ɉ Ʉ ȞrecB recC߰ɁɉĂഊວ೨ࠓȢߤ౯Ȧ൥ȧȩฝ ݚȳɥăࢍڮཱဌ༯ᇙɈڤฤ౯ɉĂrecB recC߰ Ɂ ৐ ௫ ȱĂsbcB sbcC߰ Ȧ ວ ૪ ɅsbcB sbcCɁ Ȝ ɥ ௲ ਗ Ʌ ູ Ʌ ৐ ௫ ȳ ɥăʯˁʑʷʡထ౿Ʌڽ௵ɬࡧȭȳȭɂ ȦȜɥă

recD ExoVɈʀʅʕʤʇ˄ɺĜʔߤ౯Ȧज़ഹ

ȱɀȞɥȦĂ೨ࠓȢߤ౯ɉ৐௫ȱɀȞ ɥărecD߰Ɂɉ́Ɉࢍڮཱဌ༯ᇙɬထ౿

ݢ༑ɁȜɥăʯˁʑʷʡɈထ౿Ʌڽ௵ɬ ࡧȭȳȭɂȦȜɥă

recF ʯˁʑʷʡ࠰Ɉഊວ೨ࠓȢȦज़ഹȱɀȞ ɥă

recJ ʯˁʑʷʡ࠰Ɉഊວ೨ࠓȢȦज़ഹȱɀȞ ɥă

relA1 ɺʷʦબࡪތɅൎȳɥܾຒෂɈppGppਗ

౰༑ɬज़ഹȱɀȞɥăATPćGTP3Ō-ʬ˅

ʳʑʭʁʠˁˋʑʭɿˁĜʔĪEC2.7.6.5ī ɬज़ഹȱɀȞɥă

rfbD ࡙༑โɄTDP-ˁʸʦĜʑਗ౰ਊ೧Ȧज़ ഹȱɀȤɤĂ੧ၲ࿍ჶOৣঈɬਗ౰ȳɥ ȭɂȦɁȧɄȞă

rpoH lonɅݡȢɀȭɈ༂ʏʿʛʇ๖ଖۚાɈज़

ഹɅɢɤĂʑʠ˄ʑɅɢɤᄾຩȯɦɥʯ

˅ʞɺĜʔɈཡএɬज़ഹȯȵɥărpoH am supCts߰ɁɉĂȜɥହɈʗˋʩʇଋ Ɉ਒ݗɁɈڤฤ౯Ȧ৐௫ȳɥĪhtpRɂȱ ɀɜඪɣɦɀȞɥīă

sbcB ʀʅʕʤʇ˄ɺĜʔ I ߤ౯Ȧज़ഹȱɀȞ ɥărecB recC sbcB߰ɉĂฆ௵sbcCɁ ɜȜɥăȭɈɢȠɄહ୭ໃ೓ဿڽൌɉ೨ ࠓȢ༑Ȧ਒ȩẮɈࢍڮཱဌ༯ᇙɬထ౿

ݢ༑ɁȜɥăʯˁʑʷʡɈထ౿Ʌڽ௵ɬ ࡧȭȳȭɂȦȜɥă

sbcC ฆ௵recB recC sbc BɂɂɜɅॽ୸ȯɦ ɥȦĂsbcCɈəɈ߰ɉ೨ࠓȢ༑Ȧ਒ȩĂ

́ȤɢɍʯˁʑʷʡɈࢍڮཱဌ༯ᇙɬڤ ฤโɅထ౿ݢ༑ɁȜɥă

sulA ȭɈۆ๙ાɈဿڽɉ੧ၲယᇜɬᅦ౫ȱĂ Lonဿڽ߰ȦDNAɈഹ஥ȥɣޑဌȳɥȭ ɂɬݢ༑ɅȳɥĪsuppressor of Lonīă supC(ts) ݗ๱ࠐି౯ʙ˅ʏˋ೾໴ʂĜʃĜĪUAAī

ɂɺˋʨĜĪUAGīʍʯ˄ʛʍĜɬ။ᄵ ȳɥăʢˋʓˋʑဿڽൌɉĂฝݗɁɈə ᅦ౫ȯɦɥăএ੭ɁɉtyrT^{ɂঘɊɦɀȞ} ɥă

supE ʈ˃ʗʷˋ೾໴ɺˋʨĜĪUAGīʍʯ

˄ ʛ ʍ ĜtRNAɬ ။ ᄵ ȳ ɥă Ȝ ɥ ହ Ɉ ʭɹĜʐʱʇʗĜɈཡۋɅྼᅟɁȜɥă এ੭ɁɉglnV^{ɂঘɊɦɀȞɥă} supF ʙ˅ʏˋ೾໴ɺˋʨĜĪUAGīʍʯ˄ʛ

ʍĜtRNAɬ။ᄵȳɥắgt11ɄɃɈS7 ɘȹɉS100l ́ ʭɹĜʐɈᅗސโ౺ۋɅ

ྼᅟɁȜɥăএ੭ɁɉtyrT^{ɂঘɊɦɀȞ} ɥă

thi - 1 ʙɺʷˋĪʫʗʷˋB1īਗ౰༑Ȧज़ഹȱ ɀȞɥă

traD FۚાɈ૵জ๙ൿ༑ᆵȦ൥ȧȩฝݚȱɀ

Ȟɥă

tsp ᅗࣳষɅĂݿ௰ཡএȯɦȹယྙʗˋʩʇ ଋĂɘȹɉ੧ၲଋ໘ʗˋʩʇଋɬယސȳ ɥɂ৽ȢɣɦɀȞɥɔ˂ʯˁʒʸĆʯ˅

ʞɺĜʔĪ੧ၲ့ɂ੧ၲႸɈ࠰Ʌശ੭ȳ ɥīȦज़ഹȱɀȞɥăএ੭Ɂɉprc^ɂঘ ɊɦɀȞɥă

tsx T6ʭɹĜʐࠐಹɅൎȱɀ൏౯ɬ૲ȳă

tyrT supC^ĂsupF^ɬઞ௎ă

ung ɾˁʏ˃N-ʈ˂ʋʏˁĜʔߤ౯Ȧज़ഹȱ ɀȞɥăDNAɅଲɤਮɘɦȹɾˁʏ˃ȦĂ Ung⁺ ɅɢȽɀଲɤதȥɦĂܶࡊज़ഹင ڮɬ౺Ȳɥădut ɬઞ௎ă

xyl ʅʏ˅Ĝʑɬൣଟȳɥ༑ᆵȦज़ഹȱɀȞ ɥă

(P1) P1ʯ˅ʭɹĜʐɬ။ۚȱɀȤɤĂP1౫

ঔʏʑʞʸɬཡএȳɥă

(P2) P2ʯ˅ʭɹĜʐɬ။ۚȱɀȤɤĂRed⁺ Gam⁺ ́ Ʌൎȳɥೊ်ĪSpi^– ೊ൰īȦݢ

༑ɂɄɥă

(̋80) lambdoidʯ˅ʭɹĜʐφ80ɬ။ᄵȱɀȞ ɥăȜɥହɈ߰ɉlac M15Ȧज़ഹȱɀȞ ɥdefectiveĪज़࠳īφ80ʯ˅ʭɹĜʐɬ

။ۚȱɀȞɥă

(Mu) Mu ʯ˅ʭɹĜʐăMudɉMuʭɹĜʐȦ ज़ഹȱɀȞɥȭɂɬڷჍȳɥă T7gene1

recC

322

テク ニ カ ル リ フ ァ レ ン ス

12

ドキュメント内 201314年カタログ New England Biolabs Japan (ページ 47-54)