Cloning 을위한모든것 2014-06-18 다카라코리아바이오메디칼
Contents Cloning Restriction Enzyme General Restriction Enzyme QuickCut Restriction Enzyme Cloning Restriction Enzyme/Ligation TA Cloning In-Fusion Cloning TaKaRa MiniBEST Series
Cloning Clone 세포나개체의증식에의해서생긴유전적으로동일한세포군 Cloning 목적 DNA 단편을원하는 plasmid vector에삽입 유전자공학실험의가장기본이되는기술
Cloning Flow Chart Restriction Enzyme 처리 (Restriction site 포함경우 ) Genomic DNA PCR Total RNA mrna Reverse Transcription cdna 합성 Target DNA/cDNA ( 말단에 da 포함경우 ) Target DNA/cDNA (In-Fusion Primer 로 PCR 증폭경우 )
Restriction Enzyme
Restriction Enzyme 특징 DNA 분자내의특정한염기서열을인식 dsdna의서로연결되어있는결합 (phosphodiester 결합 ) 을절단 인식서열이나그주변을 sticky/blunt end로절단 Star 활성 DNA에대하여과량의효소사용시다른서열을절단하는현상 글리세롤농도로인한 star 활성을억제하기위하여 total volume의 10% 이하로효소사용
Restriction Enzyme Takara Restriction Enzyme Unit 단위로판매 ( 일반제품, 고농도제품 ) 1 μg of λdna를절단할수있는효소양 (37, 1 hr) 8~20 U/ μl ( 고농도 : 30~60 U/ μl ) 100종의다양한 restriction enzyme 판매 효소활성에최적화된 5종 universal buffer 반응시간 30-65, 1 hr 샘플종류에따라반응시간조절 Takara Cut-Site Navigator 샘플내 restriction enzyme site 확인
QuickCut Restriction Enzyme
신상 Restriction Enzyme!! QuickCut restriction enzyme (7 월런칭예정 ) EcoR I 등베스트셀러로구성된 37 종 Single buffer 도입 Enzyme size(rxns) QuickCut Afa I 50 QuickCut Afl II 100 QuickCut Alu I 50 QuickCut Apa I 200 QuickCut BamH I 500 QuickCut Bgl II 100 QuickCut Bln I 25 QuickCut Cla I 50 QuickCut Dpn I 50 QuickCut Dra I 200 QuickCut EcoR I 500 QuickCut EcoR V 200 QuickCut Hae III 200 QuickCut Hha I 100 QuickCut Hind III 500 QuickCut Hinf I 200 QuickCut Hpa I 50 Enzyme size(rxns) QuickCut Kpn I 200 QuickCut Mlu I 100 QuickCut Nco I 25 QuickCut Nde I 200 QuickCut Nhe I 25 QuickCut Not I 25 QuickCut Pst I 500 QuickCut Pvu I 25 QuickCut Pvu II 200 QuickCut Sac I 100 QuickCut Sac II 50 QuickCut Sal I 200 QuickCut Sfi I 100 QuickCut Sma I 100 QuickCut SnaB I 25 QuickCut Spe I 25 QuickCut Sph I 50 Enzyme size(rxns) QuickCut Stu I 100 QuickCut Xba I 500 QuickCut Xho I 500 Components QuickCut Enzyme 10 QuickCut Buffer 10 QuickCut Green Buffer
신상 Restriction Enzyme!! 빠르게 cutting 하는 QuickCut Speed 편리함 기존의반응시간 (1 hr) 보다빠른 5-15 min Single buffer를이용하여사용편리성증대 기존제품과달리동일한 buffer (single buffer) 를사용하므로편리하게 double digestion 가능 BSA 추가필요없어편리 Dye 포함되어반응후 loading 빠르게 ~
Restriction Enzyme 추천 Takara Restriction Enzyme 종류 100 종 37 종 샘플적용 첨가양 고농도샘플 (>1 ug) 상관없이사용가능 샘플 1 ug 당 1 U ( 일반적으로 1 rxn 당 1 ul 로사용 ) QuickCut Restriction Enzyme <1 ug 샘플적용가능 (Linear DNA, plasmid DNA) <0.2 ug 샘플적용가능 (PCR product) 1 rxn 당 1 ul 사용 Buffer 종류 Loading dye 미포함 Loading dye 미포함 / 포함 반응조건 30-65, 1 hr 37, 5-15 min Target 고객 고농도샘플이용고객 다양한 restriction enzyme 사용고객 Cloning 고객 <200 ng 이하로사용 (Vec/insert 1:3 기준 ) MCS restriction enzyme 대다수커버 Double digestion 시 buffer 선택용이 빠른반응시간으로 cloning 시간단축
Cloning_ Restriction Enzyme/Ligation
Restriction Enzyme/Ligation Cloning 제한효소특이적 Vector 내제한효소사용 T4 DNA ligase Primer 5 말단에제한효소인식서열을부가 2개제한효소사용 방향성가진 cloning 가능, self ligation 방지 반응후정제진행 TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Code 9762A)
Ligation 추천제품 기존의 T4 DNA ligase보다월등한 DNA Ligation Kit <Mighty Mix>(Code 6023) 간편한사용 Ligation Mix를 DNA용액 (5 10 ul) 과 1 : 1 혼합 Vector : insert = 25 fmol: 25-250 fmol 5 분만에 cloning 반응! 표준모드 : 16, 30 min 고속모드 : 25, 5 min Blunt end ligation 에도최적 Sticky end, blunt end ligation, TA cloning 에서도높은효율 Component Ligation Mix 5 150 μl 1반응당 7.5 ul 사용시 100회
Ligation 제품 Code 제품명 반응시간 특징 6021 DNA Ligation Kit Ver.1 16 C, 30 min DNA 말단구조에따라반응속도가달라지는점을개선하고신속간편하게반응 6022 DNA Ligation Kit Ver. 2.1 16 C, 30 min Ver.1이 modification 된것으로적은양의 reactant volume 을사용하도효과적으로반응 DNA Ligation Kit 16 C, 30 min 6023 <Mighty Mix> 25, 5 min Ligation Mix를 DNA용액과혼합, 반응후 transformation 6024 DNA Ligation Kit LONG 긴단편의 DNA ligation에적합 16, 3 15 hr 10 kbp 이상의단편을이용시유용 6025 DNA Blunting Kit 16 C, 30 min T4 DNA polymerase의 5 3 polymerase 활성과 3 5 exonuclease 활성을이용하여 DNA 말단을평활화 Mighty Cloning Reagent Set (Blunt 6027 End) 16 C, 1 hr A-taining 된 PCR 산물을평활화시킨후 ligation 2011A T4 DNA ligase 16, 1 24 hr 가장일반적인 DNA Ligase 2160A E. coil DNA ligase 16, 1 24 hr 평활말단은 polyethylene glycol (PEG) 존재시만가능
Cloning_ TA Cloning
TA Cloning T T A T-vector Ligation A PCR product 를직접 cloning Taq polymerase A-tailing 특성이용 이용목적 PCR product 염기서열을간단히확인할경우 PCR product양이너무적을경우 Primer 제작시제한효소부위를생성하지않을경우
TA Cloning 추천제품 간편한 TA Cloning을더간편하게 Mighty TA-cloning Kit (Code 6028) pmd20-t vector 포함 단시간반응으로도높은 cloning 효율, ligation mix로간편한사용 (Code 6023 포함 ) Components (20 회 ) pmd20-t vector (50 ng/μl) Ligation Mighty Mix Positive Control Insert 20 μl 50 μl 2 10 μl T-vector pmd20 의구조 T-vector pmd19 의구조
TA Cloning Kit 제품 Code 제품명특징구성 6028 Mighty TA-cloning Kit 6019 Mighty TA-cloning Reagent Set for PrimeSTAR 3270 T-Vector pmd20 3271 T-Vector pmd19 (Simple) Mighty TA-cloning Kit 간편하게사용하는 2x ligation premix pmd20-t vector (50 ng/μl) 20 μl 16 C, 30 min Ligation Mighty Mix 50 2 μl Positive Control Insert 10 μl Mighty TA-cloning Kit PrimeSTAR 시리즈에의해증폭된 PCR A-overhang mixture 산물을 TA-cloning에이용하는경우사용 A-overhang enzyme 10 μl 간편하게사용하는 2x ligation premix 10 Buffer 20 μl datp 10 μl Vector pmd20 (50 ng/μl) 20 ul Control Insert (50 ng/μl) 10 ul T-Vector pmd19 (Simple) (50 ng/μl) 20 ul Control Insert (50 ng/μl) 10 ul
Cloning_ In-Fusion Cloning
기존 Cloning 방법 VS In-Fusion Cloning 제한효소 /ligation TA Cloning In-Fusion Cloning Cloning Vector, insert 한정 Vector 내제한효소만사 T-vector 만사용가능 Sub cloning 없이목적 용가능 vector 도입 Insert 내동일제한효소 존재시사용불가 Frame shift ( 염기가추가되면서단백질이변화되는현상 ) 제한효소추가염기로인한 추가 A 염기로인한 가능성제로!! 방향성 Double digestion 시가능 불가 방향성있는 cloning Multiple cloning 여러스텝을통해가능 불가 한번에가능!!
In-Fusion Cloning 특징 Flexible Fewer steps than traditional cloning Seamless Any insert into any location within any vector ( 제한효소사이트관계 X) Insert size (50 bp - 15 kb) Insert에제한효소처리나 A-tailing, vector phosphatse, ligase 처리 X 50 15 min 한번에 multi cloning 이나 point mutation 제작가능 Get desired construct, without adding unwanted base pairs Directional Get your insert in correct orientation
빠르게 In-Fusion Cloning~ In-Fusion reaction Linearized vector Recombinant vector Day 1 Day 2
In-Fusion 원리 In-Fusion 원리 15bp Primer 3 부분 18-25bp, GC 율 =40 60 %, Tm=58-65 (forward 와 reverse 차이 4), primer dimer and hairpin structure X
In-Fusion 의핵심 -Primer Primer design tool
In-Fusion 가이드 -PCR 정확도높은 cloning 의필수요소 CloneAmp HiFi PCR Premix (code 639298) 높은정확도 : Hot start. 낮은에러율 (12 mismatched bases per 542,580 total bases) 합성속도 : 높은 priming 효율, extension (5 sec/kb) 편리함 : 2X Premix type 적용성 : GC-rich templates. 10 kb까지합성가능 10kb 이상의 insert는? PrimeSTAR GXL DNA Polymerase
In-Fusion 가이드 -Purification Multiple band Spin Columns Non-specific 이나 multiple 밴드있을때이용 타겟 band 만잘라 spin column 정제 Single band 보다효율감소가능성존재 Cloning Enhancer Template DNA나 PCR 잔여물제거역할 Single 밴드로뜰경우에사용 정제과정이없어 loss 감소 37 15min, 80 15min
In-Fusion 가이드 -linearization NucleoSpin Gel and PCR Clean-Up Kit QuickCut 처리후 linearization 여부확인 Linearized vector는 NucleoSpin Gel and PCR Clean-Up Kit (MN) 로정제 TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Code 9762A)
In-Fusion 가이드 -In-Fusion reaction ng of vector X kb size of insert kb size of vector X molar ratio of Insert vector =insert 양 구성물 첨가량 5X In-fusion HD Enzyme premix Linearized vector PCR fragment 2 ul X ul X ul 50 15 min EcoDry 제품은 37 C 15 min, 50 C 15 min DW To 10 ul In-Fusion reaction Vector: insert=1:2 (multiple cloning 의경 우도동일 )
In-Fusion 가이드 -Transformation 정제된 plasmid 이용시 transformation 효율 반응후 transformation 효율 In-Fusion 반응액 2.5 ul 사용 (50ul competent cell에 5 ul 이상금지 ) Stellar competent cell 1 x 10 8 cfu/ug Selection 100 μg/ml of ampicillin
완벽한 Cloning 으로가는 3 가지방법 Option 1 Option 2 Option 3 In-Fusion HD Cloning Plus CE In-Fusion HD Cloning Plus In-Fusion HD EcoDry Cloning Plus CloneAmp HiFi PCR Premix In-Fusion enzyme Stellar Competent cell Cloning Enhancer CloneAmp HiFi PCR Premix In-Fusion enzyme Stellar Competent cell Spin Columns CloneAmp HiFi PCR Premix In-Fusion enzyme Stellar Competent cell Spin Columns Benefits Benefits Benefits Cloning 효율상승 정제과정없어 loss 감소 Multiple bands시에도사용가능 Reaction 필요량분주 대량실험용이 /pipetting error 감소 RT 보관가능
In-Fusion 제품 제품코드제품명용량 639648/ 639649/ 639650 In-Fusion HD Cloning Kit 10/ 50/ 100 회 639642/ 639643/ 639644 In-Fusion HD Cloning Kit w/competent Cells 10/ 50/ 100 회 639689/ 639690/ 639691 In-Fusion HD EcoDry Cloning Kit 8/ 8 3/ 96 회 639678/ 639679/ 639680 In-Fusion HD EcoDry Cloning Kit w/competent Cells 8/ 8 3/ 96 회 638909/ 638910/ 638920/ 638911 In-Fusion HD Cloning Plus 10/ 50/ 96/ 100 회 638916/ 638917/ 638919/ 638918 In-Fusion HD Cloning Plus CE 10/ 50/ 96/ 100 회 638912/ 638913/638914/ 638915 In-Fusion HD EcoDry Cloning Plus 8/ 24/ 48/ 96 회
TaKaRa MiniBEST Series
MiniBEST 를기억하라 ~ TaKaRa MiniBEST 시리즈 분류 Product Name Code Size RNA TaKaRa MiniBEST Universal RNA Extraction Kit 9767A 50 Plasmid TaKaRa MiniBEST Plasmid Purification Kit 9760A 50 PCR Clean-up TaKaRa MiniBEST DNA Fragment Purification Kit 9761A 50 Gel Extraction TaKaRa MiniBEST Agarose Gel DNA Extraction Kit 9762A 50 gdna TaKaRa MiniBEST Universal Genomic DNA Extraction Kit 9765A 50 신제품 5 종 Mini BEST 작지만강하다! 분류 Product Name Code Size Bacteria TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 9763A 50 gdna Blood TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit 9781A 50 Plant TaKaRa MiniBEST Plant Genomic DNA Extraction Kit 9768A 50 RNA Plant TaKaRa MiniBEST Plant RNA Extraction Kit 9769A 50 RNA/DNA TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 9766A 50
용도별 MiniBEST 샘플종류 어떤 TaKaRa MiniBEST 를쓸까? Universal Universal Genomic DNA Extraction Kit (9765A) Universal RNA Extraction Kit (9767A) Plant Plant Genomic DNA Extraction Kit (9768A) Plant RNA Extraction Kit (9769A) Bacteria Bacteria Genomic DNA Extraction Kit Ver.3.0 (9763A) Blood Whole Blood Genomic DNA Extraction Kit (9781A) Virus 기타 Plasmid Purification PCR Clean Up Gel DNA extraction Viral RNA/DNA Extraction Kit (9769A) Plasmid Purification Kit (9760A) DNA Fragment Purification Kit (9761A) Agarose Gel DNA Extraction Kit (9762A)
MiniBEST-Plant Series MiniBEST-Plant RNA (9769A) Plant tissue/cell로부터 total RNA 추출 50 mg sample 로부터 1-50 ug 추출 Lysis 과정후, 30 분내완료 조직특이적인 Lysis Protocol 일반식물조직 Polysaccharides, Polyphenols-rich 조직 MiniBEST-Plant DNA (9768A) Plant tissue로부터 DNA 추출 50 500 mg sample로부터 1 10 ug 추출 조직분쇄후, 40 분내완료 Protocol I. General Plant Tissue Plant roots, stem, leaves, Less or no polysaccharide, polyphenols, fat 500 ul of Buffer HS I & 10 ul of 50X DTT Buffer Weigh and grind plant tissue in liquid nitrogen Take the tissue powder the tube Protocol II. Polysaccharides, Polyphenols-rich plant tissue or fat 500 ul of Buffer HS II Weigh and grind plant tissue in liquid nitrogen Take the tissue powder the tube
MiniBEST-Bacteria and Whole Blood MiniBEST-Bacteria DNA (9763A) Gram (+), Gram (-) bacteria 로부터 DNA 추출 종특이적인 Lysis Protocol 1.0 5.0 x 10 9 sample 로부터 1-20ug 추출 Lysis 과정후, 20 분내완료 MiniBEST-Whole Blood DNA (9781A) Whole blood sample로부터 DNA 추출 Pig: 1ml fresh sample로부터 10ug 추출 Fish: 5ul fresh sample로부터 12ug 추출 동결유무에상관없이추출가능 무핵적혈구 (mammalian), 유핵적혈구 (bird, fish) 사용가능 60분내완료 Gram (-) bacteria Buffer GL Gram (+) bacteria: Lysozyme + Buffer BS + Buffer GL
MiniBEST-Viral RNA/DNA MiniBEST-Viral RNA/DNA (9766A) 다양한샘플로부터추출 다양한 sample (plasma, whole blood, virus stock solution, infected tissue) 로부터 virus RNA/DNA추출 1.0 x 10 4 copies of Hepatitis A virus 1.6 x 10 5 copies of the Flury strains of rabies virus Lysis 과정후, 20 분내완료 Sample: RNA was extracted from 200 μl of cultured cell (HL60), 10 μl of whole blood, 200 μl of saliva, 200 μl of urine, 10 mg of mouse liver Method: detected by RT-PCR with TaKaRa One Step RNA PCR Kit (AMV)