201314年カタログ New England Biolabs Japan

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ʇ˅ĜʣˋʈଌঅɈ௢੧ɉʞʇʣʃ˃˂ʭɹ ˄ˋʑĪ270ʲĜʐġīɬȮઞ௎ȩȺȯȞă ■_ޱሄ

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ˁɼʊĜʏʿˋཱܾɈʓʛʠɺʛʯ ɈʙʽĜʠ˂ɺ˃຦ކĪܑাīɬ༯ ఒෂĪNEBStickTogether.comī

DNA

ୌఄਊ೧ɂʇ˅Ĝʣˋʈࡳ୹

ယા౺ဘߔɉ౺ჩݨߔɅȤȫɥෂఘယᄘɁȜɤĂॸ࢞੮ᆧɬ੼ɥȹɛɅਊ೧โȤɢɍݛߔโɄ߇બ

ഀ੼ɬᅙȞɀȞɘȳăෂɁɜDNAୌఄਊ೧ɉഽȩɈଌঅɅێཾโɅ઴ᅙȯɦɀȞɘȳăۆ๙ા౫

সɈॸ࢞ɈȹɛɈ˄ʵĜʗĜۆ๙ાɈ৭භĂཡএސ಑ɞۆ๙ાʶʛʬˋʈɈȹɛɈˁʱ˃ݛʯ˅Ĝ ʮɈ੼౿ĂʗˋʩʇଋཡএɈȹɛɈ˂ʋˋʫʢˋʠʯˁʑʷʡɒɈۆ๙ાʇ˅ĜʣˋʈĂ૫౦ൣ ʏĜʉˋʍĜɄɃɈސ಑ࡳ୹ɈȹɛɈʍˋʯ˃෮౿ɄɃɅɉĂʅʢĜʔĂ˂ʄĜʔĂୌဌਊ೧Ăʳ

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਒࿚ଋɄ૚ᄞɬอࢷȱɀȞɘȳăȤࢊᅕɈˇĜʇʭ˅Ĝɬ஑ɛȥɣ੕ষɘɁࠊ೔Ʌ჊ȹȳɢȠɅĂ

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STEPS

TO

O

L

S

TRANSFORMATION

Competent cells (cloning & protein expression)

PR

ODUCT INFO

Polymerases AMPLIFICATION

Deoxynucleotides

Chapter 2 Chapter 8

cDNA DNA (genomic,

plasmid) STARTING MATERIAL

Reverse transcriptases & ribonucleotides

RNA

Chapters 2 & 6

CLONING TOOLS

Restriction enzymes

Kinases Ligases

Phosphatases

Chapters 1,2 & 3

SCREENING/ ANALYSIS

Restriction enzymes

Chapters 1 & 5 Ladders (DNA, RNA,

protein)

Polymerases (blunting)

Gibson Assembly™

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3

DNA

ୌఄਊ೧

ŕ

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DNA

Ligases

DNA Ligase Selection Chart 100

T4 DNA Ligase 101

Quick Ligation Kit 101

Instant Sticky-end Ligase Master Mix 102

Blunt T/A Ligase Master Mix 102

ElectroLigase 102

T3 DNA Ligase 103

T7 DNA Ligase 103

Taq DNA Ligase 103

E. coli DNA Ligase 104

9°N DNA Ligase 104

RNA Ligases 176-177

Thermostable 5´ AppDNA/RNA Ligase 177

Nucleases

Properties of Exonucleases and Endonucleases 105 Common Apps for Exonucleases & Endonucleases 106

DNase I (RNase-Free) 106

Exonuclease I 106

Exonuclease T 106

Exonuclease III 107

Lambda Exonuclease 107

Exonuclease V (RecBCD) 107

RecJ_f 108

Mung Bean Nuclease 108

Nuclease BAL-31 108

T7 Exonuclease 109

T5 Exonuclease 109

Micrococcal Nuclease 109

DNA Repair Proteins

Properties of DNA Repair Enzymes 110 DNA Repair Glycosylases on Various Damaged Bases 111

APE 1 112

T7 Endonuclease I 112

Endonuclease III (Nth) 112

Tma Endonuclease III 113

Endonuclease IV 113

Tth Endonuclease IV 113

Endonuclease V 114

T4 PDG (T4 Endonuclease V) 114

Endonuclease VIII 115

hAAG 115

Fpg 115

hOGG1 116

Uracil-DNA Glycosylase (UDG) 116

Uracil Glycosylase Inhibitor 116

Antarctic Thermolabile (UDG) 116

Afu Uracil-DNA Glycosylase (UDG) 117

hSMUG1 117

PreCR Repair Mix 89

Topoisomerase I (E. coli) 117

DNA Gyrase (E. coli) 118

DNA Gyrase (E. coli) Substrate 118

Recombinases

Cre Recombinase 118

pLox2+ (linearized) 118

DNA

Methyltransferases

CpG Methyltransferase (M.SssI) 119

GpC Methyltransferase (M.CviPI) 119

M.AluI 120

M.BamHI 120

dam Methyltransferase 120

M.EcoRI 120

M.HaeIII 120

M.HhaI 121

M.HpaII 121

M.MspI 121

M.TaqI 121

G9a Methyltransferase 250

Human DNA (cytosine-5) MTase (Dnmt1) 251

Human PRMT1 Methyltransferase 250

SET7 Methyltransferase 251

DNA

Kinases

T4 Polynucleotide Kinase 122

T4 Polynucleotide Kinase (3´ phosphatase minus) 122

DNA Phosphatases and Sulfurylases

Antarctic Phosphatase 123

Alkaline Phosphatase (CIP) 123

Pyrophosphatase, Inorganic (E. coli, yeast) 170 Thermostable Inorganic Pyrophosphatase 124

Apyrase 124

ATP Sulfurylase 124

Single-stranded DNA Binding Proteins

RecA, RecA_f 125

Tth RecA 125

T4 Gene 32 Protein 125

ET SSB (Extreme Thermostable ssBinding Protein) 126

Synthetic

Biology

BioBrick Assembly Kit 126

Gibson Assembly Master Mix 127

Cloning and Mutagenesis

Gibson Assembly Cloning Kit 127

Q5 Site Directed Mutagenesis Kit 128

Quick Blunting Kit 129

USER Enzyme 129

Cloning Plasmids and DNAs

LITMUS 28i 130

pBR322 130

pUC19 130

M13mp18 DNA 130

Lambda DNA 130

Lambda DNA (N6_{-Methyladenine-free) 130}

φX174 DNA 130

Other

β-Agarase I 131

M13KO7 Helper Phage 131

= ˂ʋˋʫʢˋʠਊ೧

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(4)

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(5)

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ɈˁɼʊĜʏʿˋȤɢɍT / Aʇ˅ĜʣˋʈĪ3Ō

Aໃ୸Ⴢ඘ɈˁɼʊĜʏʿˋīɅູݛȱȹ˂ʄĜ

ʔĆʶʑʗĜʷʛʇʑɁȜɥă်ູɄ༯ਗɅɢ ɤˁɼʊĜʏʿˋৌᆎɬྦᄠโɅ৐௫ȱɀȞ

ɥăଆݗɁ15ယ࠰ɈɼˋʅʽʱĜʏʿˋɬ਄Ƚ

ȹষĂ౼౿ɞࡏଫɄȱɅȷɈɘɘʉʷʃ˃Ćʋ ˋ ʬ ʞ ˋ ʠ ʓ ˃ Ʌ व ଋ ๖ ࠓ Ɂ ȧ ɥă ɘ ȹ ʶ ʑ

ʗĜʷʛʇʑᅗܕɉ–20ŎɈ။ശෂɅȤȞɀɜᅗ

ܕ௻ൔɁȜɥȹɛĂ઴ᅙ૪ɅᅃސɈྼᅟɉɄȞă

ཱܾ௸ॣć10 ̂lɈཱܾᅗܕෂĂ1X Blunt / TA

Ligase Master MixɂDNAɬ਼ਗă10 ̂lɈཱܾ ᅗ ܕ ෂ Ʌ1,800 cohesive end unitɈT4 DNA Ligaseɬ࠸ɚă

ʹʺćႨ౿࿚ɉ–20ŎɁᅗܕ௻ൔɁȜɥă–70Ŏ

ɅȤȫɥ๸य़ᅃސ૚অɈय़ݪĂ20ޑɈ๸य़ᅃސ

ɬ਄ȽɀɜɕɂɭɃߤ౯ɉฝݚȱɄȞȭɂɬߊ ໻ȱɀȞɥă

်ູอࢷ࿚ć

Blunt/TA Ligase Master Mix with NEB 10-beta Competent E.coli

#E9367S 1 set . . . .¥ 32,000

■_{ိߦჂ඘ɈˁɼʊĜʏʿˋ}

■_{T / A}_{ʇ˅ĜʣˋʈĪ}₃_Ō_A_{ໃ୸Ⴢ඘Ɉ} ˁɼʊĜʏʿˋī

ElectoroLigase

™

#M0369S 50 reactions . . .¥ 12,500 ತცćElectroLigaseɉʀ˄ʇʠ˅ʵ˄Ĝʏʿˋ

ɅູݛȱȹDNA˂ʄĜʔɁȜɥăPEGɬ࠸ɘɄ

ȞȹɛĂˁɼʊĜʏʿˋᅗܕɈංܶɞ౼౿ɉێ ಜ࿤ᅟɁȜɤĂȷɈɘɘʀ˄ʇʠ˅ʵ˄Ĝʏʿˋ

Ʌ઴ᅙȳɥȭɂȦɁȧɥăȳɓɀɈDNAჂ඘Īໃ

୸ĂိߦĂT / AīɬଆݗɁ30 - 60ယ࠰Ɂ਒ৌᆎ

Ɂ ˁ ɼ ʊ Ĝ ʏ ʿ ˋ ȳ ɥ ȭ ɂ Ȧ Ɂ ȧ ɥă ɘ ȹ ˂

ʄĜʔȤɢɍʨʛʭɹĜɉ–20ŎɈ။ശෂɅȤȞ

ɀɜᅗܕ௻ൔɁȜɥȹɛĂ઴ᅙ૪ɅᅃސɈྼᅟ ɉɄȞă

ཱܾ௸ॣć11 ̂lɈཱܾᅗܕෂĂ1X ElectroLigase

Reaction BufferɂDNAĂ1 ̂lɈElectroLigaseɬ

਼ਗȱĂ25ŎɁ30ယ࠰ɼˋʅʽʱĜʏʿˋă

ʹʺćႨ౿࿚ɉ–20ŎɁᅗܕ௻ൔɁȜɥă

်ູอࢷ࿚ć

ElectroLigase™ with NEB 10-beta Electrocompetent E.coli

#E9369S 1 set . . . .¥ 32,000

■_{ʀ˄ʇʠ˅ʵ˄ĜʏʿˋɅɢɥवଋ๖}

ࠓɅ੕ไ

■_{ໃ୸Ⴢ඘ĂိߦჂ඘Ă}₃_Ō_A_{ໃ୸Ⴢ඘Ī}_T

/ Aʇ˅ĜʣˋʈīɈˁɼʊĜʏʿˋ

102 DN

A修飾酵素＆

ク

ロ

ー

ニ

ン

グ

(6)

T3 DNA Ligase

#M0317S 100,000 units . . .¥ 12,000 #M0317L 750,000 units . . .¥ 48,000

ತცćT3 DNA Ligaseɉʨʇʞ˂ʂʭɹĜʐT3

ᄻᅰɈATPگശ౯Ɉ2Ⴈ੍DNA˂ʄĜʔɁȜɥă

T3 DNA LigaseɅɢɤိߦჂ඘ɞໃ୸Ⴢ඘Ɉˁ

ɼʊĜʏʿˋĂʣʛʇୌဌɬ਒ৌᆎɅ਄Ƞȭɂ ȦɁȧɥăိߦჂ඘ɈˁɼʊĜʏʿˋɬ਄Ƞ௲

ਗĂPEG6000ɬ๒ݡȳɥȭɂɁৌᆎȦ৐௫ȯɦ

ɥăT4 DNA LigaseɅ྘ɓɀĂT3 DNA Ligase

ɉ਒ȞNaCl൏౯Ī2༰īȦȜɤĂDNA˂ʄĜʔ

ߤ౯ȦྼᅟɄin vitroယા౺ဘߔʯ˅ʠʋĜ˃Ʌ ဍঢ়ȩ઴ᅙɁȧɥă

ʫʢˋʠۆ๙ાɬᄵȳɥ൥෬ࣳă

ཱܾ௸ॣć1X T3 DNA Ligase Reaction Buffer.

Į66 mM Tris-HClĂ1 mM ATPĂ10 mM MgCl2Ă 1 mM dithiothreitolĂ7.5% PEG 6000ĪpH 7.6

@25Ŏīįă25ŎɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉĂ1 X T3 DNA Ligation

Reaction Bufferɬ࠸ɚ೔ཱܾᅎᆲ20 ̂lෂĂ25ŎĂ 1ယ࠰ɅȤȞɀĂ100 ngɈ́DNA HindIII-digested

Ɉ50%ɬˁɼʊĜʏʿˋȳɥȹɛɅྼᅟɄਊ೧ᆲ

ɂȱɀฤࡹȯɦɥă

༏๱ć3,000,000 units/ml

ʹʺćATPȦྼీɁȜɥă

T3 DNA LigaseɉPEG6000ɬ ࠸ ɘ Ʉ Ȟ1X T3 DNA Ligation Reaction BufferʨʛʭɹĜෂɅȤ

Ȟɀɜߤ౯ɬ૲ȳă୒༏๱1 mMɈATPɬ๒ݡȳ

ɥăȹȺȱȭɈʨʛʭɹĜෂɁɉ1/10ڬݚɈߤ

౯ɂɄɥȭɂɅ෉ڷȳɥă਒༏๱ɈNaClɬ࠸ɚ

௸ॣɁཱܾɬ਄ȠྼᅟȦȜɥ௲ਗĂPEG6000ɬ

໴ɦɄȞၫȦᆯȞă

T3 DNA Ligaseɬ࠸ɚཱܾᅗܕɉPEGɬ࠸ɘɄ

Ȟ௲ਗɅɉ65ŎĂ10ယ࠰Ɂ࿤ߤ౯ݛɁȧɥăȹ

ȺȱPEGɬ࠸ɚ௲ਗɅɉवଋ๖ࠓȦ೫ޮȯɦɥ

ȹɛĂ༂Ʌɢɥ࿤ߤ౯ݛɬ਄ɩɄȞă

ઞ৽ဦॷć www.nebj.jpɬઞ௎

■_{ໃ୸Ⴢ඘ȜɥȞɉိߦჂ඘ɈˁɼʊĜ}

ʏʿˋ

■൏ܶ౯ɬ৐௫

■₂Ⴈ੍_DNAෂɈʣʛʇୌဌ

T7 DNA Ligase

#M0318S 100,000 units . . .¥ 12,000 #M0318L 750,000 units . . .¥ 48,000

ತცćT7 DNA Ligaseɉʨʇʞ˂ʂʭɹĜʐT7

ᄻᅰɈATPگശ౯Ɉ2Ⴈ੍DNA˂ʄĜʔɁȜɥă

2Ⴈ੍DNAෂɈᆿಞȱȹ5Ō˂ˋબɂ3Ōʪʡ˅ʅ

ʏ˃ʈ˃ĜʯɈ࠰Ʌʳʑʳʐʀʑʞ˃य़ਗɬव

౰ȳɥཱܾɬఌ༲ȳɥăT7 DNA Ligaseɉໃ୸

Ⴢ඘ɈˁɼʊĜʏʿˋȤɢɍʣʛʇୌဌɬ਒ৌ

ᆎɁ਄ȠȭɂȦɁȧɥăȱȥȱɄȦɣĂT4ɞT3

DNA LigaseɂɉڽɄɤĂိߦჂ඘ɈˁɼʊĜ

ʏʿˋɬ਄ȠȭɂȦɁȧɄȞăȷɈȹɛĂိߦ

Ⴢ඘ɂໃ୸Ⴢ඘ɬᄵȳɥDNAȦ਼੭ȳɥෂɁĂ

ໃ୸Ⴢ඘ȺȫɬˁɼʊĜʏʿˋȳɥ௲ਗɅ੕ไ ɁȜɥă

ᄻᅰćT7 DNA Ligaseۆ๙ાɬʋĜʡȳɥ˂ʋ

ˋʫʢˋʠۆ๙ાɬᄵȳɥ൥෬ࣳă

ཱܾ௸ॣć1X T7 DNA Ligase Reaction Buffer.

Į66 mM Tris-HClĂ1 mM ATPĂ10 mM MgCl2Ă 1 mM dithiothreitolĂ7.5% PEG 6000ĪpH 7.6

@25Ŏīįă25ŎɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉĂ1 X T7 DNA Ligation

Reaction Bufferɬ࠸ɚ೔ཱܾᅎᆲ20 ̂lෂĂ25ŎĂ 30ယ ࠰ Ʌ Ȥ Ȟ ɀĂ100 ngɈ́DNAɈHindIII௉ ݛ

દဘɈ50%ɬˁɼʊĜʏʿˋȳɥȹɛɅྼᅟɄ

ਊ೧ᆲɂȱɀฤࡹȯɦɥă

༏๱ć3,000,000 units/ml

ʹʺćATPȦྼీɁȜɥă

T7 DNA LigaseɉPEG6000ɬ ࠸ ɘ Ʉ Ȟ1X T7 DNA Ligation Reaction BufferʨʛʭɹĜෂɅȤ

Ȟɀɜߤ౯ɬ૲ȳă୒༏๱1 mMɈATPɬ๒ݡȳ

ɥăȹȺȱȭɈʨʛʭɹĜෂɁɉ1/10ڬݚɈߤ

౯ɂɄɥȭɂɅ෉ڷȳɥă

T7 DNA Ligaseɬ࠸ɚཱܾᅗܕɉPEGɬ࠸ɘɄ

Ȟ௲ਗɅɉ65ŎĂ10ယ࠰Ɂ࿤ߤ౯ݛɁȧɥăȹ

ȺȱPEGɬ࠸ɚ௲ਗɅɉवଋ๖ࠓȦ೫ޮȯɦɥ

ȹɛĂ༂Ʌɢɥ࿤ߤ౯ݛɬ਄ɩɄȞă

■_{ໃ୸Ⴢ඘ȺȫɬˁɼʊĜʏʿˋ}

Thermus aquaticus

Ī

_Taq

ī

_{DNA Ligase}

#M0208S 2,000 units . . .¥ 13,500 #M0208L 10,000 units . . .¥ 54,000

ತცćTaq DNA˂ʄĜʔɉĂഊၒโɄʗĜʊʛ

ʠDNA Ʌʧɼʮ˂ʘɼʒȱɀȞɥ2ȾɈᆿಞȳ

ɥʂ˂ʌʤʇ˄ʂʙʡɈ5Ō˂ˋબჂ඘ɂ3Ōʪʡ

˅ʅʏჂ඘ɂɈʳʑʳʐʀʑʞ˃य़ਗव౰ɬఌ ༲ȳɥăʂ˂ʌʤʇ˄ʂʙʡȦഊၒโʗĜʊʛ

ʠDNA ɂࠊ೔ɅൎɁȜɤĂȥȾʆʻʛʯȦɄȞ

૪ɅɈəˁɼʊĜʏʿˋȦࡧȭɥăȭɈູ౯ɬ

ᆀᅙȱɀ1ܶࡊ඲ࠓɬॳ୸ȳɥȭɂȦݢ༑ɁȜ

ɥăTaq DNA˂ʄĜʔɉ਒ݗĪ45Ŏ - 65ŎīɁ

ߤ౯ɬᄵȳɥă

ᄻᅰćThermus aquaticus HB8ȥɣʇ˅Ĝʣˋʈ ȱȹ˂ʄĜʔۆ๙ાɬཡএȳɥ൥෬ࣳȥɣ౼౿ă

ཱܾ௸ॣć1X Taq DNA Ligase Reaction Buffer Į20 mM Tris-HClĪpH 7.6@25ŎīĂ25 mM potassium acetateĂ10 mM magnesium acetateĂ10 mM DTTĂ1 mM NAD, 0.1% Triton X-100įă45ŎɁɼˋʅʽʱĜʏʿˋă

ၒۚાɂȱɀNAD+ɬྼᅟɂȳɥăNAD+ɉ10X

Taq DNA Ligase Reaction BufferɅ࠸ɘɦɀȞ

ɥăNAD+_{Ɉ཰ঌࡖɬܤෳȳɥȹɛĂʨʛʭɹĜ}

ɉ–70ŎɁ။ശȳɥă

࿚ଋࠣᆅɬȤȭɄȽɀȞɥă௢੧ɉɾɿʮʍɼ

ʠ౿࿚௢੧ɬઞ௎Īwww.nebj.jpīă

ʾʣʛʠฤࡹĪCohesive End Unitīć1 unitɉĂ

೔ཱܾᅎᆲ50 ̂lෂĂ45ŎĂ15ယ࠰ɅȤȞɀĂ1

̂gɈ́DNAɈBstEII௉ݛદဘɈ12ܶࡊൎໃ୸Ⴢ

඘Ɉ50%ɬˁɼʊĜʏʿˋȳɥȹɛɅྼᅟɄਊ

೧ᆲɂȱɀฤࡹȯɦɥă

༏๱ć40,000 units/ml

■_{൏༂౯˂ʄĜʔ}

103 DN

A修飾酵素＆

ク

ロ

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ニ

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グ

(7)

E. coli

DNA Ligase

#M0205S 200 units . . .¥ 10,500 #M0205L 1,000 units . . .¥ 42,000

ತცćᆿಞȱȹ2Ⴈ੍DNAɈໃ୸Ⴢ඘Ɉ5Ō˂ˋ

બჂ඘ɂ3Ōʪʡ˅ʅʏჂ඘ɂɈʳʑʳʐʀʑʞ

˃य़ਗɈव౰ɬఌ༲ȳɥă

ᄻᅰćʇ˅ĜʣˋʈȯɦȹE. coli DNA Ligaseۆ

๙ાɬᄵȳɥ൥෬ࣳȥɣ౼౿ă

ཱܾ௸ॣć1X E. coli DNA Ligase Reaction Buffer Į30 mM Tris-HClĪpH 8.0īĂ4 mM MgCl2Ă1

mM DTTĂ 26 ̂M NAD+Ă50 ̂g/ml BSAįăˁ

ɼʊĜʏʿˋɈ੕ไݗ๱ɉ16Ŏă

༂Ʌɢɥ࿤ߤ౯ݛć65ŎĂ20ယ࠰

ʾʣʛʠฤࡹć1 unitɉĂ೔ཱܾᅗܕ20 ̂lෂĂ

16ŎĂ30ယ࠰ɁĂHindIIIɁಜඣȱȹ́DNAඣ၀ Ī5ŌჂ ඘DNA༏ ๱ Ȧ0.12 ̂MĂĮ300 ̂g/mlįī

Ɉ50%ɬˁɼʊĜʏʿˋȳɥȹɛɅྼᅟɄਊ೧

ᆲɂȱɀฤࡹȯɦɥă

ʹʺćrATPɬྼᅟɂȳɥ഼Ɉ˂ʄĜʔɂɉڽɄ

ɤĂNAD+Īʣʋʙˋɺʷʡɺʟʣˋʐʤʇ˄

ʂʙʡīɬၒۚાɂȱɀྼᅟɂȳɥă

ိߦჂ඘ඣ၀ɈˁɼʊĜʏʿˋৌᆎɉ࣡ɛɀฝ Ȟ ȹ ɛĂ ိ ߦ Ⴢ ඘ ඣ ၀ Ɉ ˁ ɼ ʊ Ĝ ʏ ʿ ˋ Ʌ ɉ Blunt / TA Ligase Master MixĪNEB #M0367ī ȥQuick Ligation KitĪNEB #M2200īɈ઴ᅙɬ ైயȳɥă

■_{Okayama and Berg cDNA}_ʇ˅Ĝ ʣˋʈ

9 ŋ

_N

™

DNA Ligase

#M0238S 2,500 units . . .¥ 13,500 #M0238L 12,500 units . . .¥ 54,000

ತცć9ŋN™_{DNA Ligase}ɉഊၒโɄʗĜʊʛʠ

DNAɅʧɼʮ˂ʘɼʔĜʏʿˋȱɀȞɥ2ȾɈᆿ

ಞȳɥʂ˂ʌʤʇ˄ʂʙʡɈ5Ō˂ˋબჂ඘ɂ3Ō

ʪʡ˅ʅʏჂ඘ɂɈʳʑʳʐʀʑʞ˃य़ਗɈव ౰ɬఌ༲ȳɥăႨ˂ʄĜʔɉ਒ݗɁߤ౯ɬ૲ȳ Ī45Ŏ - 90Ŏīă

ᄻᅰćޠᅖ౯෰৕༂౯ɺĜʅɺɁȜɥThermococcus

sp.Īstrain 9ŋNīȥɣʇ˅Ĝʣˋʈȱȹ˂ʄĜʔ ۆ๙ાɬᄵȳɥ൥෬ࣳȥɣ౼౿ă

ཱܾ௸ॣć 1X 9ŋN DNA Ligase Reaction Buffer Į10 mM Tris-HCl, 600 µM ATPĂ2.5 mM MgCl2Ă

2.5 mM dithiothreitolĂ0.1% Triton X-100ĪpH 7.5

@ 25Ŏīįă45ŎɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹĪCohesive End Unitīć1 unitɉĂ

೔ཱܾᅗܕ50 ̂lෂĂ45ŎĂ15ယ࠰ɁĂ1 ̂gɈ ́DNAɈBstEIIɈ௉ݛદဘɈ12ܶࡊໃ୸Ⴢ඘Ɉ

50%ɬˁɼʊĜʏʿˋȳɥȹɛɅྼᅟɄਊ೧ᆲ

ɂ ȱ ɀ ฤ ࡹ ȯ ɦ ɥă Ʉ ȤCohesive End Unitɉ Nick-closing UnitɂວຑɁȜɥăʾʣʛʠɺʛʓ ɼ௸ॣɉwww.nebj.jpɬઞ௎ă

ʹʺć12ܶࡊɈʂĜʨĜˁʛʯɉ਒ৌᆎɁˁɼ

ʊĜʏʿˋȳɥȦĂ4ܶࡊɈ඗ȞʂĜʨĜˁʛʯ

ĪํळโɄ౫ঔਊ೧௉ݛඣ၀īɉˁɼʊĜʏʿˋ ȱɄȞȭɂɅ෉ڷȳɥă

■_{਒ݗݚɅȤȫɥ}_DNA_{ɈʣʛʇୌဌȦ}

ݢ༑

■_{਒Ȟ൏༂౯ɬ૲ȳȹɛĂ}_PCR_௸ॣݚ

Ɂɜ઴ᅙݢ༑

■_{Ligase Detection Reaction}_ɞ_Ligase

Chain ReactionɅȤȫɥൎᆏۆ๙ા

ູڽโۆ๙ાɈॳ୸Ȧݢ༑

■_PCR_{ഝဍෂɅ˂ˋબݛʂ˂ʌʤʇ˄}

ʂʙʡɬଲɤਮɘȵɥȭɂɅɢɥဿڽ ຩ໴Ȧݢ༑

T4 RNA Ligases

௢੧ɉ176 - 177ʲĜʐɬઞ௎

T4 RNA Ligase 1 (ssRNA Ligase) #M0204S 1,000 units . . . .¥ 10,500 #M0204L 5,000 units . . . .¥ 42,000

T4 RNA Ligase 2 (dsRNA Ligase) #M0239S 150 units . . .¥ 13,500 #M0239L 750 units . . . .¥ 54,000

T4 RNA Ligase 2, truncated

#M0242S 2,000 units . . . .¥ 11,500 #M0242L 10,000 units . . .¥ 46,000

T4 RNA Ligase 2, truncated KQ #M0373S 2,000 units . . .¥ 12,000 #M0373L 10,000 units . . . .¥ 48,000

Thermostable 5´ AppDNA/RNA Ligase #M0319S 10 rxns . . .¥ 12,000 #M0319L 50 rxns . . . .¥ 48,000

104 DN

A修飾酵素＆

ク

ロ

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(8)

Properties of Exonucleases and Endonucleases

ਊ೧ ၫ৐

DNAࡊଋ 5Ō˂ˋબ

ɬ࠸ɘɄȞ ɂȧɈߤ౯

DNAɈ߂ငڮɅ ൎȳɥ੼ᅙ1

ć

ငယಜඣĂ ێႨ੍఑ෳ2

౺౰ဘ3 Ⴢ඘࿈ૻ

FAMಜඣ4

ʳʑʳ˅ʙ ʂʀĜʠ

ಜඣ5

2̂ǩʂ˂ʌ

90%ယސɈ

ʾʣʛʠౘ6

ss ds 5Ōext 3Ōext blunt nick

Lambda Exo 5´→ 3´ +/– + +/–7 _+/– ₊ ₊ _– _3´ _{ssDNA, dNMP} ₊ _– ₂

T5 Exo 5´→ 3´ + + + + + + + NA dNMP to 6-mer + NA 2

T7 Exo 5´→ 3´ +/– + + +/– + + + 3´ ssDNA, dNMP,

dinucleotide8 + – 10

Exo III 3´→ 5´ +/– + + + +/– + + 5´ ssDNA, dNMP + – 10

Exo V both + + + + + + – Both 5´, 3´ short oligos + NA NA

RecJf 5´→ 3´ + – + +/–

9 _– _+/–10 _– _NA _dNMP _+/– _– _{> 1500}

Exo I 3´→ 5´ + – + – +/–9 _+/–10 _NR _NA _{dNMP, dinucleotide}11 ₊ _– ₂₀

Exo T 3´→ 5´ + – + – +12 _+/–10 _NR _NA _dNMP ₊ _– _>100

BAL-31 Nuclease 3´→ 5´, endo13 ₊ ₊ ₊ ₊ ₊ ₊ ₊ _NA _{dsDNA, dNMP} _NA _NA _NR

Mung Bean Nuclease endo + – + + + – – NA ssDNA, dsDNA NA NA 10

DNase I endo + + NA NA NA NA NA NA

ssDNA, dsDNA oligonucleotides, di- and trinucleotides

NA NA 0.1

Micrococcal

Nuclease endo + + NA NA NA NA NA NA

ssDNA, dsDNA 3´ monophospho- and diphosphonucleotides14

NA NA NR

T7 Endonuclease I15 _endo _– ₊ _NA _NA _NA _NA _+/– _NA _dsDNA _NA _NA _NA

௫Ɉ࿍ɉ߂ʤʇ˄ɺĜʔɈʄɼʡˁɼˋɂȱ ɀ઴ᅙɁȧɥăɄȤĂ࿍ෂɅɉၡਠȯɦɀȞ ɥȳɓɀɈߤ౯ɞູ౯Ȧࡥ੪ȯɦɀȞɥɩȫ Ɂ ɉ Ʉ Ȟă ଌ ੫ Ʌ ɉ ਊ ೧ ɞ ࡊ ଋ Ɉ ᆲĂ ɼ ˋ ʅʽʱĜʏʿˋ૪࠰ɄɃȦଌঅय़ݪɅ൥ȧȩ ܆ࣗɬᅇȢɥȭɂȦȜɥă

࿍ɈႪᇉć

+ ߤ౯ᄵɤĈ੕ไɄࡊଋ

- ঄ුɄߤ౯ɉჟȱ

+/- ੕ไɄࡊଋɂ྘ߏȱɀߤ౯Ȧฝݚ

NR ၡਠჟȱ

NA ޶ຎჟȱ

ss 1Ⴈ੍ ds 2Ⴈ੍

ext ໃ୸Ⴢ඘

dNMP ʟʂʅʏ˂ʴʤʇ˄ʂʏʡێ˂ˋબ

෉ଫć

1. ඗Ȟໃ୸Ⴢ඘ɂိߦჂ඘Ʌൎȳɥߤ౯Ʌ

ࡊȿȞɀਊ೧ɬယᇆăȭɦɣɈჂ඘ɉ౫ ঔਊ೧Ɂಜඣȱɀ੼౿ȱȹࠤڹโɄɜɈ

ɁȜɥăॳ௠Ʌ઴ᅙȱȹ5ŌȤɢɍ3Ōໃ୸

Ⴢ඘Ɉෳȯɉ4ܶࡊɁȜɥă

2. Lambda ExonucleaseĂT7 Exonuclease Ȥ ɢ ɍExonuclease IIIɅ ɢ ɥ2Ⴈ ੍DNA

ɈငယโɄಜඣɅɢɤĂ1Ⴈ੍ໃ୸Ⴢ඘ɬ

૩Ⱦ2Ⴈ੍DNAȦદ౺ȯɦɥăࠊ೔Ʉಜ

ඣɈ௲ਗĂ1Ⴈ੍DNAȦદ౺ȯɦɥă

3. ੕ไࡊଋɬࠊ೔Ʌݡ౉ယސȱȹɂȧɈદ

ဘɬࡥ੪ȳɥă

4. 5Ōɜȱȩɉ3ŌჂ඘Ʌʭ˃ʂ˄ʓɼˋࡊȦ

य़ਗȯɦȹDNAࡊଋĪ1Ⴈ੍ɜȱȩɉ2੍ī

ɈჂ඘Ʌʀʅʕʤʇ˄ɺĜʔߤ౯Ȧ੼ᅙ ȳɥȥྏȥɬ૲ȳăʳʑʳɺʷʘɼʠݛ

ߔཱܾɬᅙȞɀFAMࡊɬ૩Ⱦʂ˂ʌʤʇ

˄ʂʙʡɬਗ౰ȱȹăڬݚɈʂ˂ʌʤʇ

˄ʂʙʡɅ5ŌFAMɬ࿥ݡȱȹăȳɄɩȻĂ

ĮĪ3Ō, 6Ō- d i p i v a l o y l f l u o re s c e i n y lī - 6 - c a r b o x a m i d o h e x y lį l O -Ī2-cyanoethylī-N,N-diisopropylī– phosphoramiditeɁȜɥă3ŌFAMʂ˂ʌʤ ʇ˄ʂʙʡɉڬݚɬᅙȞɀਗ౰ȱȹăȳɄ ɩ ȻĂ1-dimethoxytrityloxy-3-Į O-ĪN-carboxy-Īdi-O-pivaloyl-fluoresceinī

-3-aminopropylīį -propyl-2-O-succinoyl-long chain alkylamino-CPGɁȜɥă

5. ʳʑʳ˅ʙʂʀĜʠय़ਗɈSp ᆏൌڽ౯ൌ

ɉĂഽȩɈʤʇ˄ɺĜʔɅɢɥಜඣɬࣅ

ȩ೫ޮȳɥȭɂȦඪɣɦɀȞɥȦĂRp ᆏ

ൌڽ౯ൌɉ೔ȩ೫ޮȱɄȞȥĂɩȴȥȱ ȥ೫ޮȱɄȞăວຑ྘Ɉᆤڽ౯ൌɬ࠸ɚ ਗ౰ʂ˂ʌʤʇ˄ʂʙʡɈ௲ਗĂಜඣɬ ʮ˅ʛʇȳɥȹɛɅထౘɈʳʑʳ˅ʙʂ

ʀĜʠȦྼᅟɂɄɥă6ᇦറɈʳʑʳ˅ʙ

ʂʀĜʠɬ࠸ɚʂ˂ʌʤʇ˄ʂʙʡɬࡊ

ଋɂȱɀॳບȱȹɂȭɧĂT7ʀˋʡʤʇ

˄ɺĜʔĪᄝ10œɈယސīɬதȩȳɓɀ

Ɉʀˋʡʤʇ˄ɺĜʔɬৌݪโĪ5%ڬݚ

ɈယސīɅʮ˅ʛʇȱȹăʀʅʕʤʇ˄

ɺĜʔɉʳʑʳ˅ʙʂʀĜʠɬ1Ⱦɘȹɉ

2ᇦറɁ࠸ɚʂ˂ʌʤʇ˄ʂʙʡɬ঄ුɅ

ယސȱȹĪࡥ੪ჟȱīă

6. ཱܾᅗܕ10 ̂lෂĂ2 ̂MɈ35 merʂ˂ ʌ ʤ ʇ ˄ ʂ ʙ ʡĪ1Ⴈ ੍ ɜ ȱ ȩ ɉ2Ⴈ ੍

DNAīɈ90œڬ௫ɬಜඣȳɥȹɛɅྼᅟ

Ʉਊ೧ɈʾʣʛʠౘăExo TɂRecJf ɬத

ȞɀĂॳ௠ȱȹȳɓɀɈਊ೧ɉৌݪโɅ ࡊଋɬࠊ೔ɅಜඣȳɥȭɂȦߊ໻ȯɦȹă

7. Lambda ExonucleaseɅɉ5Ō˂ˋબɬ࠸

ɚ2Ⴈ੍DNAɬᄭಪโɅယސȳɥभ৐Ȧ

ȜɥăȱȹȦȽɀĂێၫɈჂ඘Ʌ5Ō˂ˋ

બɬᄵȱĂɜȠێၫɈჂ඘ɅɅ5Ō˂ˋબ

ɬᄵȯɄȞ෸੍௻2Ⴈ੍DNAɬࡊଋɂȳ

ɥ ௲ ਗ Ʌ ɉĂ ˂ ˋ બ Ⴢ ඘ ȥ ɣ ᄭ ಪ โ Ʌ

DNAɬယސȳɥă

8. T7 ExonucleaseɅ ɢ ɥdsDNAɈ ݡ ౉ ယ ސદဘɉ஑ɛɅʐʤʇ˄ʂʙʡĂറȞɀ

dNMPɁȜɥȭɂȦၡਠȯɦɀȞɥă

9. RecJf ɉ5Ōໃ୸Ⴢ඘ɈိߦݛɅɉไȱɀ

ȞɄȞăExo Iɉ3Ōໃ୸Ⴢ඘ɈိߦݛɅɉ

ไȱɀȞɄȞăRecJf ɂExo IɈȞȴɦɜĂ

ཱ ܾ ɬ ާ ઻ ȳ ɥ ȹ ɛ Ʌ ɉĂ ౫ ঔ ਊ ೧ Ʌ ɢ Ƚ ɀ ੼ ౿ ȯ ɦ ɥ ɜ Ɉ ɢ ɤ ෳ Ȟ1Ⴈ ੍

DNAໃ୸Ⴢ඘ɬྼᅟɂȳɥă

10. DNA༯ ᇙ ɞ ʀ ʅ ʕ ʤ ʇ ˄ ɺ Ĝ ʔ ༏ ๱ Ʌ ɢȽɀĂRecJfĂExo IȤɢɍExo Tɉိߦ

Ⴢ඘ȥɣ2Ă3খɈʤʇ˄ʂʙʡɬதࢦȳ

ɥȭɂȦȜɥă

11. Exo Iɉ1Ⴈ੍DNAȥɣdNMPɬၪ୸ȳɥ ȦĂ੕ষɈݡ౉ယސʑʞʛʯɁɉʐʤʇ ˄ʂʙʡȦ౺౰ȯɦɥă

12. Exo Tɉ3Ōໃ୸Ⴢ඘ɈိߦݛɅ઴Ƞȭɂ

ɜ Ɂ ȧ ɥ ȦĂKlenowʵ ˂ ʹ ˁ Ĝ ʔ ɂ

dNTPɬ઴ᅙȳɥ௲ਗɅ྘ɓɀˁɼʊĜ

ʏʿˋݢ༑ɄိߦჂ඘ɬ૚অࡊଋȥɣદ

౺ȳɥৌᆎɉ2 - 4༰ڏȞă

13. BAL31ɉĂ1Ⴈ੍ʀˋʡʤʇ˄ɺĜʔߤ

౯ɄɣɍɅ3ŌŨ 5Ō 2Ⴈ੍ʀʅʕʤʇ˄

ɺĜʔߤ౯ɬᄵȳɥȭɂȦၡਠȯɦɀȞ

ɥȹɛĂɃɈɢȠɄ෸੍௻DNAɜBAL31

ɈࡊଋɂɄɥă

14. Micrococcal NucleaseɈယސદဘɉ3Ō˂ˋ બɬᄵȳɥă

15. T7 Endonucleaseɉ ʷ ʑ ʶ ʛ ʙ Ɉ Ȝ ɥ

DNAĂ୥૦ळDNAĂʳ˂ʟɼ৭ഢɜȱȩ

ɉယࡎɬ໻ૻȱɀಜඣȳɥăȭɦɣɅ྘

ɓĂʣʛʇɈ໴Ƚȹ2Ⴈ੍DNAɅൎȳɥ

੼ᅙɉඵȞă

105 DN

A修飾酵素＆

ク

ロ

ー

ニ

ン

グ

(9)

Common Applications for Exonucleases and Endonucleases

ɺʯ˂ʉĜʏʿˋ ైயਊ೧

3Ōໃ୸Ⴢ඘Ɉதࢦ Quick Blunting Kit

T4 DNA Polymerase* + dNTPs

5Ōໃ୸Ⴢ඘Ɉதࢦ

āāႰɛਮə āāಜඣ

Quick Blunting Kit

T4 Polymerase* + dNTPs, Klenow + dNTPs Mung Bean Nuclease

PCRষɈʂ˂ʌʤʇ˄ʂʙʡɈதࢦ Exonuclease I

ʯˁʑʷʡ෮౿૪Ɉ

ʊʦʸDNAɈதࢦ

Lambda Exonuclease

ĪLambda ExonucleaseɅ ɢ Ƚ ɀ ౺ ౰ ȯ ɦ ȹ1Ⴈ ੍ DNAɬExonuclease IɅɢɤதࢦȳɥȭɂɜݢ༑ī

T5 Exonuclease VĪRecBCDī

Ī෸੍௻1Ⴈ੍DNAĂ2Ⴈ੍DNAĂʣʛʇDNAɬယސī

Exonuclease VĪRecBCDī

Ī෸੍௻1Ⴈ੍DNAɂ2Ⴈ੍DNAɬယސī

RNA෮౿૪ɈDNAɈதࢦ DNase I

ʇ˅ʶʙˋჲܖ෺ਏĪChIPīސ಑ Micrococcal Nuclease

෸੍௻2Ⴈ੍DNAȥɣ1Ⴈ੍DNAɬ౺౰

āā5ŌŨ 3ŌɈၫ৐౯ȦྼᅟɄ௲ਗ

āā3ŌŨ 5ŌɈၫ৐౯ȦྼᅟɄ௲ਗ

āāێཾโɄᅙ๫Ɉ௲ਗ

Lambda Exonuclease Exonuclease III Lambda Exonuclease

* T4 DNA PolymeraseɉࣅᆵɄ3ŌŨ 5Ōʀʅʕʤʇ˄ɺĜʔߤ౯ɬ။ᄵȱɀȞɥă

DNase I

Ī

_RNase-Free

ī

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Exonuclease I

Ī

_{E. coli}

ī

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ತცć3ŌŨ 5ŌɈၫ৐Ʌ1Ⴈ੍DNAȥɣʤʇ˄ʂ

ʙʡɬதࢦȳɥă

ᄻᅰć൥෬ࣳNM554߰ȥɣʇ˅Ĝʣˋʈȱȹ

exo Iۆ๙ાɬᄵȳɥ൥෬ࣳȥɣ౼౿ă

ཱ ܾ ௸ ॣć1X Exonuclease I Reaction Buffer Į67 mM Glycine-KOHĪpH 9.5īĂ6.7 mM MgCl2Ă10 mM 2-mercaptoethanolįă37Ŏ Ɂ ɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉĂ37ŎĂ30ယ࠰ɁĂ10

nmolɈબݢᅗ౯ʤʇ˄ʂʙʡɬ౺દȳɥਊ೧ᆲ

ɂ ȱ ɀ ฤ ࡹ ȯ ɦ ɥă ʾ ʣ ʛ ʠ ɺ ʛ ʓ ɼ ௸ ॣ ɉ

ɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

■₃_ŌŨ₅_ŌɈ₁_{Ⴈ੍ʀʅʕʤʇ˄ɺĜʔ}

■_PCR_{દဘෂɈʯˁɼʶĜɈယސ}

Exonuclease T

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ತცćExonuclease TĪExo TīɉĂRNase Tɂ

ȱɀɜඪɣɦɥ1Ⴈ੍DNA / RNAູڽโʤʇ˄

ɺĜʔɁȜɥăʭ˂ĜɈ3ŌჂ඘ȥɣʤʇ˄ʂʙ

ʡɬ3ŌŨ 5ŌɈၫ৐ɅதࢦȳɥȭɂȦɁȧɥă

Exo TɉĂ3Ōໃ୸Ⴢ඘ɬ૩ȾDNAɞRNAȥɣိ ߦჂ඘ɬ੼౿ȳɥȹɛɅᅙȞɥȭɂɜɁȧɥă

ᄻᅰćExonuclease TɉCჂ඘ʶ˃ʠĜʑय़ਗʗˋ

ʩʇଋĪMBPīᅃਗʗˋʩʇଋɂȱɀݿ௰ཡএ

ȯɦ౼౿ăɺʭɻʣʞɻĜʇ˅ʶʠʈˁʭɻĜ

ষĂMBPȥɣಜඣȯɦȹExo Tɉ1খɈɺʷʦબ

ȦNჂ඘Ʌ࿥ݡȯɦɀȤɤĂMetȦPheɅဿࠓȯ

ɦɀȞɥĪȳɄɩȻĂMet-Exo TɁɉɄȩ

Glu-Phe-Exo TɂɄɥīă

ཱܾ௸ॣć1 X NEBuffer 4Į50 mM potassium

a c e t a t eĂ2 0 m M Tr i s - a c e t a t eĂ1 0 m M magnesium acetateĂ1 mM dithiothreitolĪpH

7.9īįă25ŎɁɼˋʅʽʱĜʏʿˋă

25ŎĂ30ယ࠰ɁĂ1 nmolɈĮ3

Hį-࿈ૻʵ˂ʙ

ʷʐˋȥɣ0.1 nmolɈTCAᅗސ౯DNAɬ౺౰ȳ

ɥȹɛɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥă

ʹʺćExo TɈRNAɅൎȳɥߤ౯ɉĂDNAɅൎ

ȳɥߤ౯ɂڽɄɥăRNAɈ௲ਗĂ࿈ஈโɄཱܾ

௸ॣݚɁ1.0 pmolɈrA20ɬࠊ೔Ʌ௉ݛȳɥȹɛ

ɅɉĂ1 unitɈExo TȦྼᅟɂɄɥă

■₁_Ⴈ੍_DNA_Ă₁_Ⴈ੍_RNA_Ʌູڽโ

■_DNA_ȜɥȞɉ_RNA_Ɉ₃_{Ōໃ୸Ⴢ඘Ɉိ}

ߦݛ

106 DN

A修飾酵素＆

ク

ロ

ー

ニ

ン

グ

(10)

Exonuclease III

Ī

_{E. coli}

ī

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ತცć2Ⴈ੍DNAɈ3Ō-ʪʡ˅ʅʏჂ඘ȥɣʺʦ

ʤʇ˄ʂʙʡɈඦިโɄதࢦɬఌ༲ȳɥăࡊଋ ɂɈय़ਗ૪Ʌதࢦȳɥʤʇ˄ʂʙʡౘȦঔɣɦ

ɀȞɥȹɛĂDNAယાɈज़଄Īယސᆲīɬ෮౿

ȳɥȭɂȦɁȧɥă

ູɅపሆ౯Ɉ਒ȞࡊଋɉိߦჂ඘ɜȱȩɉ3Ō࠳

ႣჂ඘ɁȜɥȦĂ2Ⴈ੍DNAෂɈʣʛʇɅൎȱɀ

ɜ੼ᅙȱɀ1Ⴈ੍ʆʻʛʯɬ౺౰ȳɥăႨਊ೧ɉ

1Ⴈ੍DNAɅൎȱɀߤ౯ɬ૲ȯɄȞȹɛĂ3Ō-ໃ

୸Ⴢ඘ɉಜඣȯɦɄȞăಜඣȯɦɅȩȯɉໃ୸

Ⴢ඘ɈෳȯɅگശȱĂໃ୸Ⴢ඘Ȧ4ܶࡊڬ௫ɁȜ

ɦɊ࣡ɛɀಜඣȯɦɅȩȞăȭɈɢȠɄູ౯ɬ

ᆀᅙȱɀĂಜඣȯɦɅȩȞჂ඘Ī3Ōໃ୸Ⴢ඘ī

ȤɢɍಜඣȯɦɞȳȞჂ඘Īိߦɜȱȩɉ5Ōໃ

୸Ⴢ඘īɬ૩Ⱦ෸੍ယાȥɣฤၫ৐౯Ɉज़଄ɬ ਄ȠȭɂȦɁȧɥă

ਊ೧ߤ౯ɉĂDNA৭ഢĪɒ˂ʛʇʑīɞ༯ᇙɅ

Ȝɥา๱گശȳɥĪC>A = T>GīăɘȹĂཱܾ

ݗ๱Ăܶ༏๱Ȥɢɍਊ೧ / DNA྘Ȧਊ೧ߤ౯Ʌ

൥ȧɄ܆ࣗɬ࢒ɖȳȹɛĂไࡱཱܾ௸ॣɈ੕ไ ݛȦྼᅟɁȜɥă

ɘȹĂExonuclease IIIɜĂRNase HĂ3Ō-ʳʑʭɹ

ʗĜʔȤɢɍAP-ʀˋʡʤʇ˄ɺĜʔߤ౯ɬᄵȳ

ɥȭɂȦၡਠȯɦɀȞɥă

ᄻᅰć൥෬ࣳK-12ĂBE257 / pSGR3߰ĪB.Weiss ્Ɉ৕ڷɅɢɤอࢷȯɦȹīȥɣ౼౿ă

ཱܾ௸ॣć1 X NEBuffer 1Į10 mM Bis Tris

Propane-HClĂ10 mM MgCl2Ă1 mM DTTĪpH

7.0Ă25Ŏīįă37ŎɁɼˋʅʽʱĜʏʿˋă

37ŎĂ30ယ࠰ɁĂ1 nmolɈબݢᅗ౯ʤʇ˄ʂʙ

ʡɬ౺દȳɥਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛ

ʠɺʛʓɼ௸ॣɉɾɿʮʍɼʠɬઞ௎Īwww.

nebj.jpīă

ʹʺćʳʑʳ˅ʙʂʀĜʠय़ਗɉExonuclease

IIIɅɢȽɀಜඣȯɦɄȞă˷-ʳʑʳ˅ʙʂʀĜ

ʠɬ࠸ɚʤʇ˄ʂʙʡɬଲɤਮɘȵĂێၫɈჂ ඘ɬ။ীȳɥȭɂɁฤၫ৐౯Ɉज़଄ɬ਄Ƞȭɂ ɜɁȧɥă

■₃_ŌŨ₅_{Ōʀʅʕʤʇ˄ɺĜʔ}

■_{unidirectional nested deletions}_Ɉ੼౿

■_{ငڮູڽโဿڽຩ໴}

■_{੍ູڽโɄʯ˅ĜʮɈ෮౿}

■_{ʐʟʂʅʏʏĜʉˋʏˋʈɈȹɛɈ}₁

Ⴈ੍DNAࡊଋɈ෮౿

Lambda Exonuclease

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ತცć5ŌŨ3ŌɈၫ৐Ʌ2Ⴈ੍DNAȥɣ5Ōʺʦʤ

ʇ˄ʂʙʡɈதࢦɬఌ༲ȳɥ਒Ȟʯ˅ʓʛʏ ʫʞɻĜɬ૩Ⱦਊ೧ɁȜɥăపሆ౯Ɉ਒Ȟࡊଋ

ɉ5Ō-˂ˋબݛ2Ⴈ੍DNAɁȜɥȦĂ1Ⴈ੍Ȥɢɍ

˂ˋબݛȯɦɀȞɄȞࡊଋɜฝȞཱܾബ๱ɄȦ ɣယސȳɥăႨਊ೧ɉĂʣʛʇɞʆʻʛʯȥɣ

DNAɬယސȳɥȭɂɉɁȧɄȞă

ᄻᅰć൥෬ࣳᄻᅰˁʸʘʀˋʡʤʇ˄ɺĜʔɂ

ʶ˃ʠĜʑय़ਗʗˋʩʇଋĪMBPīɈᅃਗʗˋ

ʩʇଋɬɺʭɻʣʞɻĜʇ˅ʶʠʈˁʭɻĜɅ

ɢ ɤ ౼ ౿ ȱ ȹ ষĂLambda Exonucleaseȥ ɣ

MBPɬಜதȱɀ౼౿ă

ཱ ܾ ௸ ॣć1X Lambda Exonuclease Reaction

BufferĮ67 mM Glycine-KOHĪpH 9.4@25ŎīĂ 2.5 mM MgCl2Ă50 ̂g/ml BSAįă37ŎɁɼˋ ʅʽʱĜʏʿˋă

37ŎĂ30ယ࠰ɁĂ10 nmolɈબݢᅗ౯ʟʂʅʏ

˂ʴʤʇ˄ʂʙʡɬ2Ⴈ੍ࡊଋȥɣ౺౰ȳɥȹɛ

ɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛʠɺʛ

ʓɼ௸ॣɉɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

ʹʺć5Ōʪʡ˅ʅʏჂ඘Ɉယސബ๱ɉĂ5Ō˂ˋ

બჂ඘Ʌ྘ɓɀ20༰ඵȞă1Ⴈ੍DNAɈယސബ

๱ɉĂ2Ⴈ੍DNAɅ྘ɓɀ100༰ඵȞă

■_{਒Ȟʯ˅ʓʛʏʫʞɻĜɬᄵȳɥ}₅_Ō

Ũ 3Ōʀʅʕʤʇ˄ɺĜʔ

■₂_Ⴈ੍_DNA_ȥɣ₅_{Ōʺʦʤʇ˄ʂʙʡɬ}

தࢦ

Exonuclease V

Ī

_RecBCD

ī

#M0345S 1,000 units . . .¥ 12,000 #M0345L 5,000 units . . .¥ 48,000

ತცćExonuclease Vɉ൥෬ࣳᄻᅰɈRecBCD

ထਗൌɁȜɤĂATPگശ౯1Ⴈ੍DNAʀˋʡʤʇ

˄ɺĜʔߤ౯ɞĂ1Ⴈ੍Ȥɢɍ2Ⴈ੍DNAʀʅʕ

ʤʇ˄ɺĜʔߤ౯ɬ࠸ɚࡐȾȥɈڽɄɥߤ౯ɬ

ᄵȱɀȞɥăȷɦȸɦɈݡ౉ယސɉ2ၫ৐ȥɣᇦ

റโɅࡧȭɤĪ3ŌჂ඘Ȥɢɍ5ŌჂ඘ɈᆤၫȥɣīĂ

ʂ ˂ ʌ ʤ ʇ ˄ ʂ ʙ ʡ ɬ ౺ દ ȳ ɥă ȳ ɓ ɀ Ɉ

Exonuclease VɈߤ౯Ʌɉ2ݟɈʃʙʂˋɬྼీ

ɂȳɥăʀʅʕʤʇ˄ɺĜʔߤ౯ɅɉMg2+ Ȧྼ

ీɁȜɥĂCa2+ _{ɉʀʅʕʤʇ˄ɺĜʔߤ౯ɬ೫}

ޮȱĂݡ౉ယސɬࡧȭȯȴɅ2Ⴈ੍DNAɬɕɃȩ

Īʰ˂ʃĜʔߤ౯īȭɂȦɁȧɥă

ᄻ ᅰć൥ ෬ ࣳ ᄻ ᅰ ɈExonuclease VɈ ʍ ʮ ʾ

ʣʛʠɁȜɥRecBĂRecCĂRecDɬཡএȳɥȹ

ɛɈʯˁʑʷʡɬᄵȳɥ൥෬ࣳă

ཱܾ௸ॣć1X NEBuffer 4Į50 mM potassium

a c e t a t eĂ2 0 m M Tr i s - a c e t a t eĂ 1 0 m M magnesium acetateĂ1 mM dithiothreitolĪpH

7.9īįă37ŎɁɼˋʅʽʱĜʏʿˋă

37ŎĂ30ယ࠰ɁĂ2Ⴈ੍DNAȥɣȥɣ1 nmolɈ બݢᅗ౯ʟʂʅʏ˂ʴʤʇ˄ʂʙʡɬ౺౰ȳɥ ȹɛɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥă

■_෸੍௻₁_Ⴈ੍_DNA_Ȥɢɍ₂_Ⴈ੍_DNA_ɬ

ယސ

■_{ʣʛʇȦຩ໴ȯɦȹʯˁʑʷʡɂʑĜ}

ʩĜʋɼ˃௻ʯˁʑʷʡɉယސȱɄȞ

107 DN

A修飾酵素＆

ク

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ー

ニ

ン

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(11)

RecJ

f

#M0264S 1,000 units . . .¥ 12,000 #M0264L 5,000 units . . .¥ 48,000

ತცćRecJf ɉ5ŌŨ 3Ōၫ৐ɅDNAȥɣʟʂʅʏ

ʤʇ˄ʂʙʡێ˂ˋબɈதࢦɬఌ༲ȳɥ1Ⴈ੍

DNAູڽโʀʅʕʤʇ˄ɺĜʔɁȜɥăRecJf

ɉĂᅗސ౯ɬ৐௫ȯȵɥȹɛɅRecJɂʶ˃ʠĜ

ʑय़ਗʗˋʩʇଋĪMBPīɈᅃਗʗˋʩʇଋɂ

ȱɀཡএȯɦɥă

22ܶࡊɈ5Ōໃ୸Ⴢ඘ɬ૩ȾDNAȦRecJf Ɉࡊଋ

ɂȱɀᅙȞɣɦȹ௲ਗĂय़ݪɂȱɀိߦჂ඘Ă5Ō

ໃ୸Ⴢ඘Ī1-5ܶࡊīȤɢɍ5Ō࠳ႣჂ඘Ī1 - 8

ܶ ࡊ ī ɬ ᄵ ȳ ɥDNAඣ ၀ Ɉ ਼ ਗ ဘ Ȧ ౺ ౰ ȯ ɦ

ɥăɄȤRecJf ɉ5Ō˂ˋબɬྼᅟɂȱɄȞă

ᄻᅰćRecJf ɉCჂ඘ʶ˃ʠĜʑय़ਗʗˋʩʇଋ

ĪMBPīᅃਗʗˋʩʇଋɂȱɀݿ௰ཡএȯȵɀ౼

౿ăMBPɉRecJf Ɉఌ༲ߤ౯Ʌ܆ࣗɬᅇȢɥȭ

ɂɄȩĂRecJf Ɉݢᅗ౯ɬ৐௫ȯȵɥă

ཱܾ௸ॣć1 X NEBuffer 2Į50 mM NaClĂ10

mM Tris-HClĂ10 mM MgCl2Ă1 mM DTTĪpH 7.9 @ 25Ŏīįă37ŎɁɼˋʅʽʱĜʏʿˋă

37ŎĂ30ယ࠰ɁĂ0.05 nmolɈTCAݢᅗ౯ʟʂ ʅʏ˂ʴʤʇ˄ʂʙʡɬ౺౰ȳɥȹɛɅྼᅟɄ ਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛʠɺʛʓɼ௸

ॣɉɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

■₁_Ⴈ੍_DNA_ູڽโ₅_ŌŨ₃_Ōʀʅʕʤʇ

˄ɺĜʔ

■_DNAȥɣʐʟʂʅʏʤʇ˄ʂʙʡێ

˂ˋબɬதࢦ

Mung Bean Nuclease

#M0250S 1,500 units . . .¥ 11,000 #M0250L 7,500 units . . .¥ 44,000

ತ ცć1Ⴈ ੍DNAȤ ɢ ɍ1Ⴈ ੍RNAɅ ູ ڽ โ Ʉ ʀ ˋ ʡ ʤ ʇ ˄ ɺ Ĝ ʔ Ɂ Ȝ ɤĂDNAယ ા Ȥ ɢ ɍ

RNAယાɈ1Ⴈ੍ໃ୸ငယɬယސȱɀˁɼʊĜ

ʏʿˋݢ༑ɄိߦჂ඘ɬ౺౰ȳɥă

ᄻᅰćMung Bean sprouts

ཱܾ௸ॣć1X Mung Bean Nuclease Reaction

BufferĮ50 mM sodium acetateĪpH 5.0 at 25ŎīĂ30 mM NaClĂ1 mM ZnSO4įă30ŎɁ ɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉĂ37ŎĂ1ယ࠰ɁĂ1 ̂g

Ɉબݢᅗ౯ʤʇ˄ʂʙʡɬ౺౰ȳɥȹɛɅྼᅟ Ʉਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛʠɺʛʓɼ

௸ॣɉɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

ʹʺćਊ೧଄ߤɈ೐ɅDNAȦྥູڽโಜඣȯɦ

ɥȹɛĂ༂Ʌɢɥ଄ߤɉȱɄȞă

■₁_{Ⴈ੍ໃ୸ငယĪ}₃_ŌȤɢɍ₅_{Ōໃ୸Ⴢ඘ī}

ɬதࢦȱĂˁɼʊĜʏʿˋݢ༑Ʉိߦ Ⴢ඘ɬ੼౰

■_{๖ଖʶʛʬˋʈ}

■_{ʰɺʬˋ˃ĜʯɈಜඣ}

■_ʊʦʸ_DNA_{ȥɣۆ๙ાʋĜʡ༯ᇙɬ}

ಜத

■_{ఛȱȞ౫ঔਊ೧ငڮɈ੼౿}

Nuclease BAL-31

#M0213S 50 units . . .¥ 11,000 #M0213L 250 units . . .¥ 44,000

ತ ცćNuclease BAL-31 ExonucleaseɉĂ2Ⴈ

੍DNAɈ3ŌȤɢɍ5ŌჂ඘Ɉᆤၫɬယސȳɥă໘

င༯ᇙɉಜඣȱɄȞăႨਊ೧ɉʣʛʇɞʆʻʛ

ʯȤɢɍ2Ⴈ੍DNAɈ1Ⴈ੍ငယɞRNAɬູڽโ

Ʌಜඣȳɥ1Ⴈ੍ʀˋʡʤʇ˄ɺĜʔɁɜȜɥă

ᄻᅰćAlteromonas espejiana BAL-31Ɉ༱ᅤܕ

ȥɣ౼౿ăĶFastķȤɢɍĶSlowķळਊ೧Ɉ਼ਗ

ဘɁȜɥă

ཱܾ௸ॣć1X Nuclease BAL-31 Reaction Buffer

Į600 mM NaClĂ20 mM Tris-HClĪ25ŎĂ pH8.0īĂ1 mM EDTAĂ12 mM MgCl2Ă12 mM

CaCl2įă30ŎɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉ30ŎĂ10ယ࠰ɁĂ1X

Nuclease BAL-31 Reaction Buffer 50 ̂lෂĂ ෸ ੍ ௻2Ⴈ ੍ΦX174 DNAĪ40 ̂g/mlī Ɉ ߂ Ⴢ

඘ȥɣ200ܶࡊൎɬଲɤதȩȹɛɅྼᅟɄਊ೧ᆲ

ɂȱɀฤࡹȯɦɥă

ʹʺćႨਊ೧Ɂஐᆅȱȹ2Ⴈ੍DNAჂ඘ɉိߦჂ

඘Ȥɢɍໃ୸Ⴢ඘Ɉ਼ਗဘɁȜɥăȭɈ਼ਗဘ ɉ෸ಞʇ˅ĜʣˋʈȳɥȭɂȦɁȧɥȦĂৌᆎ

ɢȩˁɼʊĜʏʿˋȳɥȹɛɅɉĂไಜɄDNA

ʵ˂ʹˁĜʔɁໃ୸Ⴢ඘ɬୌဌȳɥྼᅟȦȜɥă

઴ᅙ෸೐ɅঔɤĂਊ೧ᅗܕɬཱܾʨʛʭɹĜɁ ࡏଫȳɥȭɂȦɁȧɥă

■₂_Ⴈ੍_DNA_{ɬᆤჂ඘ȥɣယސ}

■_{౫ঔਊ೧ငڮɈʶʛʬˋʈ}

108 DN

A修飾酵素＆

ク

ロ

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ニ

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グ

(12)

T7 Exonuclease

#M0263S 1,000 units . . .¥ 11,000 #M0263L 5,000 units . . .¥ 44,000

ತცćT7 Exonucleaseɉ5ŌŨ 3ŌɈၫ৐Ʌ2Ⴈ੍

DNAɈ5Ōʺʦʤʇ˄ʂʙʡɈதࢦɬఌ༲ȳɥă

T7 Exonucleaseɉ5ŌჂ඘ɈɕȥĂ2Ⴈ੍DNAɈ ʆʻʛʯɞʣʛʇȥɣɜʤʇ˄ʂʙʡɈதࢦɬ

ާ઻ȳɥȭɂȦɁȧɥă5Ō˂ˋબɈᄵჟɅ࠲ब

Ʉ ȩDNAɬ ယ ސ ȳ ɥă ɘ ȹRNA/DNAʧ ɼ ʮ

˂ʛʡȥɣRNAȤɢɍDNAɬ5ŌŨ 3Ōၫ৐Ʌယ

ސȳɥȭɂɜၡਠȯɦɀȞɥȦĂ2Ⴈ੍ɜȱȩɉ

1Ⴈ੍RNAɉယސȱɄȞă

ᄻᅰćTYB12ɼˋʞɼˋᅃਗʗˋʩʇଋɬᄵȳ

ɥ൥෬ࣳȥɣ౼౿ă

ཱܾ௸ॣć1 X NEBuffer 4Į50 mM potassium

a c e t a t eĂ2 0 m M Tr i s - a c e t a t eĂ1 0 m M magnesium acetateĂ1 mM DTTĪpH 7.9Ă

25Ŏīįă25ŎɁɼˋʅʽʱĜʏʿˋă

25ŎĂ30ယ࠰ɁĂ1 nmolɈબݢᅗ౯ʟʂʅʏ˂

ʴʤʇ˄ʂʙʡɬ2Ⴈ੍DNAȥɣ౺౰ȳɥȹɛɅ

ྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛʠɺʛ

ʓɼ௸ॣɉɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

■₅_ŌŨ₃_{Ōʀʅʕʤʇ˄ɺĜʔ}

■_DNA_ȥɣ₅_{Ōʺʦʤʇ˄ʂʙʡɬதࢦ}

T5 Exonuclease

#M0363S 1,000 units . . .¥ 11,000 #M0363L 5,000 units . . .¥ 44,000

ತცćT5 Exonucleaseɉ5ŌŨ 3Ōၫ৐ɅDNAɬ

ယސȳɥă5ŌჂ඘ڬެɅɜ෸੍௻ȜɥȞɉࠞ௻2

Ⴈ੍DNA௫ɈʆʻʛʯȜɥȞɉʣʛʇȥɣɜʤ

ʇ˄ʂʙʡɈதࢦɬ਄ȠȭɂȦɁȧɥăȱȥȱ

ɄȦɣĂʑĜʩĜʋɼ˃௻2Ⴈ੍DNAɉಜඣȱɄ

ȞăT5 ExonucleaseɉɘȹĂ1Ⴈ੍DNAʀˋʡ ʤʇ˄ɺĜʔߤ౯ɜᄵȱɀȞɥă

ᄻᅰćT5ʭɹĜʐᄻᅰɈD15ۆ๙ાɬ࠸ɚʯˁ

ʑʷʡɬᄵȳɥ൥෬ࣳă

a c e t a t e , 2 0 m M Tr i s - a c e t a t e , 1 0 m M magnesium acetate, 1 mM dithiothreitolĪpH 7.9@25Ŏīįă37ŎɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹć1 unitɉĂ37ŎĂ30ယ࠰ɁĂ2Ⴈ

੍DNAȥɣ1 nmolɈબݢᅗ౯ʤʇ˄ʂʙʡɬ౺

౰ȳɥȹɛɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥă ʾʣʛʠɺʛʓɼ௸ॣɉɾɿʮʍɼʠɬઞ௎ Īwww.nebj.jpīă

■_෸੍௻₁_Ⴈ੍_DNA_Ă₂_Ⴈ੍_DNA_Ȥɢɍ

ʣʛʇȦຩ໴ȯɦȹʯˁʑʷʡɬယސ

■_{ʑĜʩĜʋɼ˃௻ʯˁʑʷʡɉယސȱ}

ɄȞ

Micrococcal Nuclease

#M0247S 320,000 gel units . . .¥ 12,000 ತ ცćMicrococcal NucleaseɉStaphylococcus aureusᄻᅰɈྥູڽโʀˋʡʤʇ˄ɺĜʔɁȜ ɥăႨਊ೧ɉ˂ʋˋʫʢˋʠ൥෬ࣳȥɣ౼౿ȯ

ɦɀȤɤĂ2Ⴈ੍Ă1Ⴈ੍Ăࠞ௻Ȥɢɍ෸੍௻Ɉ

߇બɬယސȳɥăpH 7.0 - 10.0ɈཱྀڱɁߤ౯ɬ

૲ȱĂpH 9.2ɅȤȞɀRNAȤɢɍDNAɈᆤၫɅ ൎȱɀ૖ไߤ౯ɬ૲ȳăɺʟʣ˃બĂʟʂʅʏ ɺʟʣ˃બɘȹɉʙʷʐ˃બ࠸ᄵᆎȦ਒Ȟငڮ ɅൎȱɀĂᄭಪโɅಜඣȳɥȭɂȦၡਠȯɦɀ

ȞɥăDNAȤɢɍRNAɉ3Ōʳʑʳʺʦʤʇ˄ʂ

ʙʡȤɢɍʐʤʇ˄ʂʙʡɅယސȯɦɥă

ᄻᅰćMicrococcal Nucleaseۆ๙ાĪGeneIDć

3238436īɂʶ˃ʠĜʑय़ਗʗˋʩʇଋĪMBPī

ۆ๙ાɈᅃਗۆ๙ાɬ။ᄵȳɥ൥෬ࣳȥɣཡএ

ȯȵȹɈȻĂMBPɬಜதȱɀ౼౿ă

ཱܾ௸ॣć1X Micrococcal Nuclease Reaction

BufferĮ50 mM Tris-HClĪpH 7.9@25ŎīĂ5 mM CaCl2į Ʌ100 ̂g/ml BSA ɬ ๒ ݡă37Ŏ ɁɼˋʅʽʱĜʏʿˋă

ʾʣʛʠฤࡹćĪKunitzʾʣʛʠī1unitɉĂ37ŎĂ

30ယ࠰ɁĂ࢓৉๱ɬO.D.1.0Ī260 nmīഝݡȯ

ȵɥબݢᅗ౯ʂ˂ʌʤʇ˄ʂʙʡɈၪ୸Ʌྼᅟ Ʉਊ೧ᆲɂȱɀฤࡹȯɦɥă

Īɺʄ˅Ĝʑʊ˃ʾʣʛʠī1ʊ˃ʾʣʛʠɉĂ

37ŎĂ15ယ࠰ɁĂ1 ̂gɈ́ʊʦʸDNAɬယސȱĂ

ฝယાDNAඣ၀Ī100 - 400ܶࡊൎīɈරಓȦ

1.2%ɺʄ˅Ĝʑʊ˃௫Ɂ௉଄ȳɥȹɛɅྼᅟɄ

ਊ೧ᆲɂȱɀฤࡹȯɦɥă

ʹʺć10,000 Gel UnitsɉĂȤɢȷ1,000 Kunitz unitsɅഊຎȳɥă

ʾʣʛʠɺʛʓɼ௸ॣɉɾɿʮʍɼʠɬઞ௎ Īwww.nebj.jpīă

༏๱ć2 x 106

gel units/ml

ʹ ʺćߤ ౯ Ʌ ɉ1 - 5 mM Ca2+ _{Ȧ ྼ ᅟă}

100

mM჎჊Ɉܶ༏๱ɅȤȞɀĂpH 7 - 10Ɂߤ౯ɬ

૲ȳăݿ௰ᆲɈEGTAɬݡȢɥȭɂɅɢɤĂਊ೧

ɬ࿤ߤ౯ݛȳɥȭɂȦɁȧɥă

■_{ʗˋʩʇଋ෮౿૪Ɉ߇બယސ}

■_{In vitro}_Ⴉᄟ

■_{੧ၲᅗސဘɈ༈๱ɬฝঌ}

■_{ʇ˅ʶʙˋ৭ഢސ಑}

■_఻ബɄ_RNA_{ʏĜʉˋʏˋʈ}

■_ChIP_ސ಑

109 DN

A修飾酵素＆

ク

ロ

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ニ

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グ

(13)

Properties of DNA Repair Enzymes

DNAୌဌʈ˂ʋʏˁĜʔɈࡊଋ

Ȥɢɍಜඣદဘ

࿍ɈႪᇉć

AP ɺʯ˂ˋ / ɺʬ˂ʷʐˋငڮ

P ˂ˋબ

OH ʪʡ˅ʅʏ

dR5P ʟʂʅʏ˂ʴĜʑ-5Ō-˂ˋબ PA 3Ō-ʳʑʳ-˷Ă

˸-࿤ၼሆɺ˃ʟʪʡ

ਊ೧ ࡊଋ ಜඣငڮ

ཱܾɅɢɤ౺౰ȳɥჂ඘

5Ō-Ⴢ඘ 3Ō-Ⴢ඘

APE 1 APငڮ 1_ഹ஥ɈྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ

5Ōത dR5P OH

Endo III બݛʬ˂ʷʐˋĂAPငڮ 1ྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ

ഹ஥Ɉ3Ōത P PA

Endo IV AP sites 1_ഹ஥ɈྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ₅_Ō_ത dR5P OH

Endo V

ʟʂʅʏɼʦʏˋĂ

ʷʑʶʛʙĂʰɺʬˋ / ൎɅ

ɄȽɀȞɄȞ˃ĜʯĂ

ʭˁʛʯĂࡴૣY৭ഢ

2Ă3ྈᄆɈʳʑʳʐʀʑʞ˃

य़ਗഹ஥Ɉ3Ōത P OH

Endo VIII બݛʬ˂ʷʐˋĂAPငڮ 1_ഹ஥ɈྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ

5ŌȤɢɍ3ŌതɈᆤၫ P P

Fpg બݛʯ˂ˋĂAPငڮ 1ྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ

ഹ஥Ɉ5ŌȤɢɍ3ŌതɈᆤၫ P P

hOGG1 બݛʯ˂ˋĂAPငڮ 1_ഹ஥ɈྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ

3Ōത P PA

hNEIL1 બݛʬ˂ʷʐˋĂ_{બݛʯ˂ˋĂ}_AP_ငڮ 1_ഹ஥ɈྈᄆɈʳʑʳʐʀʑʞ˃य़ਗ₅_Ō_Ȥɢɍ₃_Ō_Ɉᆤၫ P P

T7 Endo I

୥૦वĂʳ˂ʟɼ ʐʻˋʇʏʿˋĂ

ʷʑʶʛʙĂʰʞ˅2Ⴈ੍

1Ă2Ă3ྈᄆɈʳʑʳʐ

ʀʑʞ˃य़ਗ

ഹ஥Ɉ5Ōത P OH

T4 PDG ʬ˂ʷʐˋʘɼʶĜ

ʘɼʶĜɈ5ŌʙʷˋതɅȜɥ

N-ʈ˂ʋʏ˃य़ਗȤɢɍ

APငڮɈ3ŌതɅȜɥ1ྈᄆɈ ʳʑʳʐʀʑʞ˃य़ਗ

P* PA

UDG

Afu UDG ʟʂʅʏɾ˂ʐˋ N-ʈ˂ʋʏ˃य़ਗ AP site (no break)

hSMUG1

ʟʂʅʏɾ˂ʐˋ

5-ʪʡ˅ʅʏɾˁʏ˃

5-ʪʡ˅ʅʏʹʙ˃ɾˁʏ˃

5-ʳ˃ʷ˃ɾˁʏ˃

N-ʈ˂ʋʏ˃य़ਗ AP site (no break)

hAAG ʟʂʅʏɼʦʏˋĂ_{ɺ˃ʅ˃ݛʯ˂ˋ} N-ʈ˂ʋʏ˃य़ਗ AP site (no break)

* ʬ˂ʷʐˋʘɼʶĜȦࢼᄵय़ਗȱɀȞɥă

110 DN

A修飾酵素＆

ク

ロ

ー

ニ

ン

グ

(14)

DNA Repair Glycosylases on Various Damaged Bases

2Ⴈ੍DNAʂ˂ʌĪ34-merī

ENZYME AP:A DHT:A 5-hmU:A 5-hmU:G I:T 6-MeA:T 8-0G:C 8-0G:G U:A U:G

THYMINE GLYCOL:A

ୌဌਊ೧

APE 1 ++++ + – – – – – – – – –

Endo III ++++ + – – – – – – – – +

Tma Endo III ++++ ++ – – – – + + – – ++

Endo IV ++++ + – – – – – – – – –

Tth Endo IV ++++ + – – – – – + – – –

Endo V* +++ + + + ++++ + ++ + + ++++ ++

Endo VIII ++++ ++ – – – – – – – – +++

Fpg + + – – – – ++++ ++++ – – +

hAAG – – – – ++++ – – – – – –

hNEIL1 ++++ ++ – – – – + + – – ++

hOGG1 ++ – – – – – ++++ + – – –

T4 PDG ++++ – – – – – – – – – –

UDG N/A – – – – – – – ++++ + –

Afu UDG N/A – – – – – – – ++++ + –

hSMUG1 N/A – +++ +++ – – – – ++++ ++++ –

ਊ೧ߤ౯ɈപฤɉൣൖܶࡊɬᅙȞɀ࿈ஈโɄཱܾ௸ॣݚɁ਄Ƚȹă * ʣʛʇɈəĂഹ஥ɬதࢦȱɄȞă

1Ⴈ੍DNAʂ˂ʌĪ34-merī

ENZYME AP DHT 5-hmU I 6-MeA 8-0G U

THYMINE GLYCOL:A

ୌဌਊ೧

APE 1 ++ – – – – – – –

Endo III ++ – – – – – – –

Tma Endo III ++ + – – – – – –

Endo IV – – – – – – – –

Tth Endo IV – – – – – – – –

Endo V + – – ++++ – + – –

Endo VIII +++ – – – – – – –

Fpg + + – – – + – +

hAAG – – – + – – – –

hNEIL1 + + – – – – – +

hOGG1 ++ – – – – + – –

T4 PDG – – – – – – – –

UDG N/A – – – – – ++++ –

Afu UDG N/A – – – – – ++++ –

hSMUG1 N/A – ++ – – – +++ –

࿍ɈႪᇉć

AP ɺʯ˂ˋ / ɺʬ˂ʷʐˋငڮăɾˁ ʏ˃ɬ࠸ɚʂ˂ʌɬUDGஐᆅȳɥ ȭɂɅɢȽɀAPငڮȦ੼౿ȯɦɥă

DHT 5,6-ʐʪʡ˅ʙʷˋ

5HMU 5-ʪʡ˅ʅʏʹʙ˃ɾˁʏ˃

I ɼʦʏˋ

6MeA 6ʹʙ˃ɺʟʣˋ

8-OG 8ʂʅʕʈɺʣˋ

U ɾˁʏ˃

APćA ɺʯ˂ˋ / ɺʬ˂ʷʐˋငڮȦɺʟ

ʣˋɂܶࡊൎɬव౰

DHTćA 5,6-ʐʪʡ˅ʙʷˋȦɺʟʣˋɂܶ

ࡊൎɬव౰

5HMUćA 5-ʪʡ˅ʅʏʹʙ˃ɾˁʏ˃Ȧɺ

ʟʣˋɂܶࡊൎɬव౰

5HMUćG 5-ʪʡ˅ʅʏʹʙ˃ɾˁʏ˃Ȧʈ

ɺʣˋɂܶࡊൎɬव౰

IćT ɼʦʏˋȦʙʷˋɂܶࡊൎɬव౰

6MeAćT 6-ʹʙ˃ɺʟʣˋȦʙʷˋɂܶࡊ

ൎɬव౰

8-OGćC 8-ʂʅʕʈɺʣˋȦʏʠʏˋɂܶ

ࡊൎɬव౰

8-OGćG 8-ʂʅʕʈɺʣˋȦʈɺʣˋɂܶ

ࡊൎɬव౰

UćA ɾˁʏ˃Ȧɺʟʣˋɂܶࡊൎɬव౰

UćG ɾˁʏ˃Ȧʈɺʣˋɂܶࡊൎɬव౰

ୌဌɈ˄ʱ˃ć

++++ 100% +++ 50% ++ 10% - 25% + < 10%

- ॳ୸ȯɦɥਊ೧ߤ౯ჟȱĪ< 0.7%ī

N/A ޶ຎჟȱ

111 DN

A修飾酵素＆

ク

ロ

ー

ニ

ン

グ

(15)

APE 1

#M0282S 1,000 units . . .¥ 12,000 #M0282L 5,000 units . . .¥ 48,000

ತცćʪʠᄻᅰɺʯ˂ˋ/ɺʬ˂ʷʐˋĪapurinic

/ apyrimidinicćAPīʀˋʡʤʇ˄ɺĜʔĪAPE

1īɉĂHAP 1ɘȹɉRef-1ɂȱɀɜඪɣɦɀȤ

ɤĂ൥෬ࣳɈExonucleaseIIIʗˋʩʇଋɂഊວ౯

ɬᄵȳɥăAPE 1ɉĂAPငڮɈ5ŌതɅȜɥʳʑ

ʳʐʀʑʞ˃ਬ߆ɬݡ౉ယސɅɢȽɀಜඣȱĂ3Ō

-ʪʡ˅ʅʏჂ඘Ȥɢɍ5Ō-ʟʂʅʏ˂ʴĜʑ˂ˋ

બჂ඘ɬ૩Ⱦ1Ⴈ੍DNAඣ၀ɬ౺౰ȳɥăɘȹ

APʀˋʡʤʇ˄ɺĜʔߤ౯Ɉ഼ɅĂଯȞDNA

3Ō-ʐʀʑʞˁĜʔߤ౯Ă3ŌŨ 5Ōʀʅʕʤʇ˄

ɺĜʔߤ౯ȤɢɍRNaseHߤ౯ɬ૩Ⱦȭɂɜၡ

ਠȯɦɀȞɥă

DNAୌဌߤ౯Ɉ഼ɅĂAPE 1ɉin vitroɅȤȞɀ

ഽȩɈ๖ଖۚાɈDNAय़ਗߤ౯ɬ෮౲ȳɥ࡙༑

ɜᄵȱɀȞɥăAPE 1ɉFos-Junʰʞ˅ʘɼʶĜĂ

Jun-JunʳʺʘɼʶĜĂHeLa੧ၲɈAP-1ʗˋʩ

ʇଋĂȯɣɅɉNF-kBĂMybȤɢɍATF / CREB

ʭɹʷ˂ĜʗˋʩʇଋɈ๖ଖۚાɈDNAय़ਗߤ

౯ɬ৐௫ȯȵɥȭɂȦၡਠȯɦɀȞɥă

ᄻᅰćʇ˅ĜʣˋʈȱȹʪʠAPE 1ۆ๙ાɬᄵ

ȳɥ൥෬ࣳă

a c e t a t eĂ2 0 m M Tr i s - a c e t a t eĂ1 0 m M magnesium acetateĂ1 mM dithiothreitolĪpH

37ŎĂ1૪ ࠰ ɁĂ1খ ɈAPင ڮ*ɬ ૩ Ⱦ20 pmol

Ɉ34 mer 2Ⴈ੍ʂ˂ʌʤʇ˄ʂʙʡɬಜඣȳɥ

ȹɛɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥă௢੧ɉ

ɾɿʮʍɼʠɬઞ௎Īwww.nebj.jpīă

* ɾˁʏ˃રࡊɬ1ܶࡊ࠸ɚ20 pmolɈ34 mer 2Ⴈ੍ʂ˂ ʌʤʇ˄ʂʙʡɬ1 unitɈUracil-DNA GlycosylaseĪUDGī Ɂ37ŎĂ2ယ࠰ஐᆅȳɥȭɂɅɢɤAPငڮɬ੼౿ă

ʋʹʛʠɺʛʓɼɅైயȯɦɥࡏଫć1ć103ă

௢੧Ʉʯ˅ʠʋĜ˃ɉwww.nebj.jpɬઞ௎ă

■_{ඍ੧ၲʊ˃๝࡜܋຦}

Īʋʹʛʠɺʛʓɼī

■ɺ˃ʃ˂ᅗ୸

■ɺ˃ʃ˂ࠆȧᄋȱ

■ޜᆯʣʛʇʠˁˋʑ˄Ĝʏʿˋ

T7 Endonuclease I

#M0302S 250 units . . .¥ 12,000 #M0302L 1,250 units . . .¥ 48,000

ತ ცćT7 Endonuclease Iɉ ʷʑ ʶʛʙ ɈȜɥ DNAĂ ୥ ૦ ळDNAĂ ʳ ˂ ʟ ɼ ৭ ഢ ɜ ȱ ȩ ɉ ယ

ࡎĂʰʞ˅2Ⴈ੍DNAɬ໻ૻȱɀಜඣȳɥăɘ

ȹĂȭɦɢɤɜཱܾബ๱ɉฝȞɄȦɣĂʣʛʇ

Ɉ໴Ƚȹ2Ⴈ੍DNAɜ໻ૻȱɀಜඣȳɥăಜඣင

ڮɉʷʑʶʛʙɈ5ŌതɅȜɥ1ྈᄆĂ2ྈᄆĂɜ

ȱȩɉ3ྈᄆɈʳʑʳʐʀʑʞ˃य़ਗɁȜɥă

ᄻᅰćʶ˃ʠĜʑय़ਗʗˋʩʇଋĪMBPīɂĂ

CჂ ඘ ʙ ʂ ʀ ʑ ʞ ˃ ɬ ૩ Ⱦ ࿤ ߤ ౯ ळT7

Endonuclease IĪtT7 Endo IīĪێငज़ഹळīɈ ᅃਗʗˋʩʇଋɂȱɀཡএȱȹɜɈɁȜɥăज़ ໵ȳɥɺʷʦબરࡊɅഊຎȳɥਗ౰ʲʯʙʡɬin vitroɁtT7 Endo Iɂᇦय़ȯȵɥȭɂɅɢɤĂࠊ

೔ෳɈߤ౯ळT7 Endo IȦ౺౰ȯɦɥă

ཱ ܾ ௸ ॣć1X NEBuffer 2Į50 mM NaClĂ10 mM Tris-HClĂ10 mM MgCl2Ă1 mM DTTĪpH

࿚ଋࠣᆅɬ਄ȽɀȞɥă௢੧ɉɾɿʮʍɼʠɬ ઞ௎Īwww.nebj.jpīă

37ŎĂ1૪࠰ɁĂ1 ̂gɈʑĜʩĜʋɼ˃୥૦ळ

pUCĪATīɬ90%ڬ௫෸੍௻ɅဿࠓȳɥȹɛɅ

ྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥăʾʣʛʠɺʛ

ʓɼ௸ॣɉwww.nebj.jpɬઞ௎ă

* pUCĪATīɉpUC19ɈEcoRIငڮɂPstIငڮɈ࠰Ʌʵ˂˂

ˋʃĜɬ೾໴ȱȹɜɈɅᄻᅰȳɥă

༂Ʌɢɥ࿤ߤ౯ݛć࿤ݢ

ʹʺćT7 Endonuclease Iɉ৭ഢೊ൰౯ɬᄵȳɥ

ਊ೧ɁȜɥăᅕęɄDNAࡊଋɅൎȱɀڽɄɥ྘

ߤ౯Ɂ੼ᅙȳɥăಜඣɬ਄ȠࡊଋȮɂɅ઴ᅙȳ ɥਊ೧ᆲɞཱܾ૪࠰ɬ੕ไݛȳɥྼᅟȦȜɥă

42Ŏڬ௫Ɂཱܾɬ਄Ƚȹ௲ਗĂྥູڽโʤʇ˄

ɺĜʔߤ౯Ȧ౺Ȳɥă

■_ʷʑʶʛʙ_DNA_ɬ໻ૻ

■_{୥૦ळɜȱȩɉယࡎ}_DNA_ɬယސ

■_{ʰ ʞ ˅}₂_{Ⴈ ੍ Ȥ ɢ ɍ ʣ ʛ ʇ Ɉ ໴ Ƚ ȹ}

DNAɬॳ୸ɘȹɉಜඣ

■_{ʏʿʛʠʄˋʇ˅ĜʣˋʈᅙɅ෸੍௻}

DNAɬˁˋʘʸɅಜඣ

Endonuclease III

Ī

_Nth

ī

#M0268S 1,000 units . . .¥ 12,000 #M0268L 5,000 units . . .¥ 48,000

ತცćEndonuclease IIIĪNthīɉ൥෬ࣳᄻᅰɈ

ʀˋʡʤʇ˄ɺĜʔɁȜɥăN-ʈ˂ʋʏˁĜʔ

ߤ౯ȤɢɍAP-˂ɺĜʔߤ౯ɬᄵȱɀȤɤĂ

N-ʈ˂ʋʏˁĜʔߤ౯Ʌɢɤ2Ⴈ੍DNAȥɣഹ஥ɬ

ିȫȹʬ˂ʷʐˋɬதࢦȱɀĂAPငڮɬ౺౰ȳ

ɥăႨਊ೧ɈAP-˂ɺĜʔߤ౯ɉAPငڮɈ3Ōത

ɬಜඣȱĂ5Ō˂ˋબȤɢɍ3Ō-ʳʑʳ-˷,˸-࿤ၼ ሆɺ˃ʟʪʡɬ౺౰ȳɥă

Endonuclease IIIĪNthīɅɢȽɀ໻ૻĆதࢦȯɦɥ

ഹ஥ܶࡊɂȱɀɉĂ໶೧Ă5, 6-ʐʪʡ˅ʅʏʙʷˋĂ

ʙʷˋʈ˂ʋĜ˃Ă5-ʪʡ˅ʅʏ-5ʹʙ˃ʪʘˋʠˋĂ

ɾˁʏ˃ʈ˂ʋĜ˃Ă6-ʪʡ˅ʅʏ-5, 6-ʐʪʡ˅

ʙʷˋĂmethyltartronylureaɄɃȦȜɥă

ᄻᅰćʇ˅Ĝʣˋʈȱȹnthۆ๙ાɬᄵȳɥ൥෬ࣳă

ཱܾ௸ॣć1X Endonuclease III (Nth) Reaction BufferĮ20 mM Tris-HClĂ1 mM EDTAĂ1 mM DTTĪpH 8.0Ă25Ŏīįă37ŎɁɼˋʅʽʱĜʏʿˋă

37ŎĂ1૪࠰ɁĂ1খɈAPငڮ*ɬ૩Ⱦ1 pmolɈ

34 mer 2Ⴈ੍ʂ˂ʌʤʇ˄ʂʙʡɬಜඣȳɥȹ

ɛɅྼᅟɄਊ೧ᆲɂȱɀฤࡹȯɦɥă

* ɾˁʏ˃રࡊɬ1ܶࡊ࠸ɚ10 pmolɈ34 mer 2Ⴈ੍ʂ˂ʌ ʤʇ˄ʂʙʡɬ1 unitɈUracil-DNA GlycosylaseĪUDGī Ɂ37ŎĂ2ယ࠰ஐᆅȳɥȭɂɅɢɤAPငڮɬ੼౿ă

ʋʹʛʠɺʛʓɼɅైயȯɦɥࡏଫć1ć104_ȥ

ɣ1ć105ă௢੧Ʉʯ˅ʠʋĜ˃ɉwww.nebj.jp

ɬઞ௎ă

■_{ඍ੧ၲʊ˃๝࡜܋຦}

Īʋʹʛʠɺʛʓɼī

■_{ɺ˃ʃ˂ᅗ୸}

■_{ɺ˃ʃ˂ࠆȧᄋȱ}

201314年カタログ New England Biolabs Japan

DNA

ୌఄਊ೧ɂʇ˅Ĝʣˋʈࡳ୹

3

DNA

ୌఄਊ೧

ŕ

ʇ˅Ĝʣˋʈ

DNA

Ligases

Nucleases

DNA Repair Proteins

Recombinases

DNA

Methyltransferases

DNA

Kinases

DNA Phosphatases and Sulfurylases

Single-stranded DNA Binding Proteins

Synthetic

Biology

Cloning and Mutagenesis

Cloning Plasmids and DNAs

Other

DNA Ligase Selection Chart

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100

DN

A修飾酵素 ＆

ク

ロ

ー

ニ

A修飾酵素＆

_Kit

A修飾酵素＆

A修飾酵素＆

_Taq

_{DNA Ligase}

A修飾酵素＆

_N