하반기 계획 (Clontech, 외 )

Transcription

1 TaKaRa MiniBEST 시리즈 -Nucleic Acid Purification Kits 다카라코리아바이오메디칼

2 Nucleic Acid Purification?

3 Nucleic Acid Purification Products Purification Kit 품질기대치 고품질 경쟁력있는가격 가격

4 TaKaRa MiniBEST 시리즈 분류 Product Name Code Size gdna TaKaRa MiniBEST Universal Genomic DNA Extraction Kit 9765A 50 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 분류 Product Name Code Size Bacteria TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver A 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 A 50

5 TaKaRa MiniBEST gdna 추출제품

6 TaKaRa MiniBEST gdna products 분류 Product Name Code Size Universal gdna TaKaRa MiniBEST Universal Genomic DNA Extraction Kit 9765A 50 Bacteria gdna TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver A 50 Blood gdna TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit 9781A 50 Plant gdna TaKaRa MiniBEST Plant Genomic DNA Extraction Kit 9768A 50

7 TaKaRa MiniBEST Universal Genomic DNA Extraction Kit(#9765) Product Description 혈액, Gram (-) Bactria, 포유류배양세포, 동물 / 식물 tissue 등 다양한시료에서 genomic DNA 추출 Spin Column membrane 과특별한 lysis buffer 를채용 용해된조직세포로부터 20 분이내전과정수행

8 실험과정 No. Components Effect 1 Buffer GL: 2 Proteinase K: lysis tissue or cell and release DNA Digest protein, enable tissue to be more easily lysis and release DNA 3 RNase A(10 mg/ml): Remove residual RNA in the lysates 4 Buffer GB: 5 Buffer WA: Make the DNA binding in the membrane Wash salt (isopropyl alcohol), contaminating proteins 6 Buffer WB: Wash salt (ethanol) 7 Elution Buffer: Elute the DNA 보관및운송 1. The kit can be stored at room temperature (15-25 ). 2. Proteinase K can be stored at room temperature (15-25 ) for 6 months, and should be stored at -20 for long-term. 3. RNase A (10 mg/ml) can be stored at room temperature (15-25 ) for 6 months, and should be stored at -20 for long-term. 4. The kit is shipped at room temperature (15-25 ).

9 Comparison of each kit for DNA extraction [Low Range PFG PFGE] 250 ng mouse spleen gdna Low Range PFG PFGE M M 1: TAKARA 9765 Extraction of DNA 97 K 48.5 K 23.1 K 9.42 K 2: O Extraction of DNA 3: Q Extraction of DNA 4: T Extraction of DNA 5: M Extraction of DNA [Conclusion] TaKaRa MiniBEST(Code 9765) 로추출핚경우 gdna 가온젂하고따라서 Genomic library 제작에더적합하다

10 Universal Genomic DNA Extraction Kit 가격비교 Product Code rxn List Price cost/rxn Takara TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver A ,000 3,040 Qiagen QIAamp DNA Mini Kit ,000 3,860 Promega Wizard Genomic DNA Purification Kit A ,000 2,460 Intron G-spin Genomic DNA Extraction Kit (for Bacteria) ,000 2,800 i-genomic BYF DNA Extraction Mini Kit ,000 3,600 Bioneer AccuPrep Genomic DNA Extraction Kit K ,000 1,480

11 TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (code 9763A)

12 Key feature and benefits Features - Gram (+), Gram (-) 를포함핚다양핚 bacteria로부터고순도 gdna 추출 - Gram positive bacteria ( 펩티도글리칸세포벽 ) 로부터 gdna 추출에적합 - Simple, convenient, high-yield DNA, good purity - Cell lysis 과정이후 20분이내에젂과정수행가능 Efficiency x 10 9 bacteria 로부터 1-20 ug 의 genomic DNA 추출가능 Applications - PCR, Southern blotting, RAPD, AFLP, RFLP, and other molecular biology experiments

13 Protocol Two Lysis protocol Gram positive, Gram negative bacteria 에따라 Lysis protocol 을선택하여적용 Components Proteinase K (20 mg/ml) Lysozyme (20 mg/ml) RNaseA (10 mg/ml) Buffer BS Buffer GL Buffer GB Buffer WA Buffer WB Elution Buffer Spin Column Collection tube Box I (stored at -20 ) Action Digestion of proteins Destruction of bacteria cell wall Decomposition RNA Box II (stored at RT (15-25 ) Cells were resuspended Lysis of bacteria release DNA DNA binding in the membrane Wash salt (isopropanol), miscellaneous protein Wash salt (ethanol) Elution DNA

14 Protocol - Treatment of different bacteria Gram-negative Bacteria Cleavage : Collect 1.0~5.0E+09 cells,the supernant was discarded Gram-positive Bacteria Cleavage : Collect 1.0~5.0E+09 cells,the supernant was discarded Buffer GL, Proteinase K and RNase A and mix thoroughly shaken, and incubated at 56 water bath for 10 minutes Join Buffer GB, mix well. The lysate was added to a Spin Column, follow the cleaning and stripping steps to continue the follow-up action. Join Buffer BS resuspended cells, adding Lysozyme, mix thoroughly shaken, and incubated at 37 for 60 minutes Centrifuged, the supernatant was discarded. Add Buffer GL, Proteinase K and RNase A, mix thoroughly shaken, and incubated at 56 water bath for 10 minutes. Join Buffer GB, mix well. Gram-positive bacteria Lysis : Buffer BS 와 Lysozyme 을이용하여 Petidoglycan 구조를분해 Petidoglycan * Lysozyme: Peptidoglycan층의 N-아세틸뮤라민산과 N-아세틸글루코사민사이의 β-1,4-글리코시드결합을가수분해

15 Experimental Example - Gram-positive Bacteria DNA extraction results Purpose :Evaluated using extraction TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 Grampositive Bacteria DNA's. Material : 2 OD Gram (+) Bacteria DNA Extraction follows Gram-positive Bacteria DNA Yield(ug) Bacillus subtilis 10 Lactobacillus 6 Acidiphilium facilis 6 Kocuria kristinae 8 OD 600 =1, equivalent to 1ml broth containing 1.0x10 9 cells The experimental results :10/200 loading M M : λ-hind III digest 1 :Bacillus subtilis genomic DNA 2 :Lactobacillus plantarum genomic DNA 3 :Acidiphilium facilis genomic DNA 4 :Kocuria kristinae genomic DNA Conclusion : Using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 good for a Grampositive Bacteria Extraction of genomic DNA.

16 Experimental Example - Gram-negative Bacteria DNA extraction results Purpose :Evaluated using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 for Gram-negative Bacteria DNA extraction results. Material : 2 OD Gram(-) Bacteria DNA extraction as follows: Gram-negative Bacteria DNA Yield(ug) Acetobacter aceti 6 Enterobacter aerogenes 8 Serratia marcescens Sb 8 E.coli JM The experimental results :10/200 loading M M : λ-hind III digest 1 :Acetobacter aceti genomic DNA 2 :Enterobacter aerogenes genomic DNA 3 :Serratia marcescens Sb genomic DNA 4 :Escherichia.coli JM109 genomic DNA OD 600 =1, equivalent to 1ml broth containing 1.0x10 9 cells Conclusion : Using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 good for a Gramnegative Bacteria Extraction of Genomic DNA

17 DNA extraction amount from different bacteria 2E +09 starting Gram-positive Bacteria that can be extracted 2E +09 initiation of Gram-negative Bacteria that can be using this kit extracted using this kit The amount of DNA as : The amount of DNA as follows: Material Microbacterium oxydans Bacillus subtilis Kocuria varians Staphylococcus aureus 3A Enterococcus durans. Pediococcus pentosaceus Enterococcus gallinarum Anabaena flos-aquae Arthrobacter luteus Nocardia Brevibacterium linens Caryophanon latum L Caseobacter polymorphus Streptomyces Lactobacillus plantarum DNA yield 6~10 μg 8~10 μg 6~10 μg 6~10 μg 6~10 μg 6~10 μg 5~8 μg 6~10 μg 6~12 μg 5~8 μg 5~8 μg 5~8 μg 5~8 μg 5~8 μg 5~8 μg Material Escherichia.coli JM109 Acetobacter aceti Acinetobacter calcoaceticus Acidiphilium organovorum 1 3H Enterobacter aerogenes Flavobacterium balustinum Plesiomonas shigelloides Serratia marcescens Sb DNA yield 6~10 μg 10~12 μg 10~15 μg 10~15 μg 6~10 μg 10~15 μg 6~10 μg 10~15 μg Note:Individual Gram-positive bacteria (eg: Bacillus thuringiensis (Bt) of cell wall structure is rather special, more difficult lysis, DNA extraction volume is slightly less.

18 minibest 가격비교 Maker Descption Code# Rxn. Price cost/rxn Memo TaKaRa Bacterial Genomic DNA Extraction Kit Ver ,000 3,160 Qiagen QIAamp DNA Mini Kit (50) ,000 4,040 Qiagen QIAamp DNA Mini Kit (250) ,000 3,664 genomic, mitochondrial, bacterial, pa rasite, or viral DNA genomic, mitochondrial, bacterial, pa rasite, or viral DNA Promega Wizard Genomic DNA Purification Kit A ,000 2,460 white blood cells, tissue culture cells, animal tissue, plant tissue, yeast and Gram-positive and Gram-negative ba cteria Intron i-genomic BYF DNA Extraction Mini Kit ,000 3,600 gram+ Bacteria, Yeast, Fungi Intron G-spin Genomic DNA Extraction Kit (for Bacteria) ,000 2,800 gram+/gram- Bacteria

19 TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit (code 9781A)

20 Key feature and benefits Features - Whole Blood sample로부터 genomic DNA 추출 - Suitable for fresh and frozen 50ul-2ml of whole blood extract - 무핵적혈구 (non-nucleated erythrocyte) 를포함핚 whole blood (mammalian) 1ul ml, 유핵적혈구 (nucleated erythrocyte) 를포함핚 whole blood (bird, fish) 의 10ul 로부터 gdna 추출가능 - Simple, convenient, high-yield DNA, good purity - 60분이내에젂과정수행가능 Applications - PCR, Southern blotting, RAPD, AFLP, RFLP, and other molecular biology experiments

21 Protocol Sample preparation -For Whole Blood with non-nucleated erythrocyte: 200 ul 1 ml -For Whole Blood with nucleated erythrocyte : no more than 10 ul Components Proteinase K (20 mg/ml) RNaseA (10 mg/ml) 10X Buffer RCL A 10X Buffer RCL B Buffer GB Buffer WA Buffer WB Elution Buffer Spin Column Collection tube Box I (stored at -20 ) Action Digestion of proteins Making it easier to cell lysis Decomposition RNA Box II (stored at RT (15-25 ) RCL A: RCL B = 1:4 mixture Lysis of Red Blood Cells DNA binding in the membrane Wash salt (isopropanol), miscellaneous protein Wash salt (ethanol) Elution DNA Whole procedures: Erythrocyte lysis, DNA binding, DNA Purification

22 Experimental Example different sources of DNA extraction result of whole blood Purpose : Evaluated using TaKaRa MiniBEST Blood Genomic DNA Extraction Kit, different sources of whole blood DNA extraction. Material : Na 2 EDTA blood anticoagulant different sources Electrophoresis results : M 1 M 2 M 3 M 4 Source Material The initial dose The yield of DNA (ug) Horse 1 ml 3 ug Pig 1 ml 10 ug Man 1 ml 5 ug Fish 5 ul 12 ug M : λ-hind III digest 1 :Horse Whole Blood 2 :Pig Whole Blood 3 :Man Whold Blood 4 :Fish (nucleated red blood cells) Conclusion : TaKaRa MiniBEST Blood Genomic DNA Extraction Kit 은다양핚종의 Whole Blood 로부터 Genomic DNA 를추출가능하다. 특히, Fish 와같은종의 nucleated Red Blood Cell 로부터도고효율의 gdna 를 추출가능하다.

23 Experimental Example - trace blood gdna extraction results gdna electrophoresis results: fresh whole blood gdna gdna electrophoresis results: frozen porcine whole blood gdna M M M : λ-hind III digest 1 :Starting 1 ul of whole blood of mice, gdna extraction results 2 :Starting 5 ul of whole blood of mice, gdna extraction results 3 :200 ul of whole blood starting mouse, gdna extraction results M : λ-hind III digest 1 :Starting 1 ul of whole blood of mice, gdna extraction results 2 :Starting 5 ul of whole blood of mice, gdna extraction results 3 :200 ul of whole blood starting mouse, gdna extraction results Conclusion : TaKaRa MiniBEST Blood Genomic DNA Extraction Kit minimum blood extraction 1ul starting genomic DNA, DNA fragments were detected by electrophoresis

24 minibest 가격비교 Maker Descption Code# Rxn. Price cost/rxn Memo TaKaRa Whole Blood Genomic DNA Extraction Kit ,000 3,040 Qiagen QIAamp DNA Blood Mini Kit (50) ,000 3,840 MN NucleoSpin Blood QuickPure(50) NucleoSpin Blood, (50) ,300 3,546 genomic, mitochondrial, or viral DNA from blood and related body fluids Promega ReliaPrep Blood gdna Miniprep System A ,000 2,850 Thermo GeneJET TM Whole Blood Genomic DNA Purificatio n Kit K ,000 3,220 Intron IQeasy Plus Blood RNA Extraction Mini Kit ,000 5,400 whole blood 에서 total RNA 추출 gdna remover spin column system 적용 (2 column spin type)

25 TaKaRa MiniBEST Plant Genomic DNA Extraction Kit (code 9768A)

26 Key feature and benefits Features - Plant tissue로부터 genomic DNA 추출 - Two unique lysis system (general plant tissue, polysaccharides, polyphenols-rich plant tissue) - Simple, convenient, high-yield DNA, good purity - Tissue ground이후, 40분이내에젂과정수행가능 Efficiency mg 식물조직으로부터 1 10ug 의고순도 genomic DNA 추출 Applications - PCR, Southern blotting, RAPD, AFLP, RFLP, and other molecular biology experiments

27 Protocol Two unique lysis system - general plant tissue (Protocol I) - polysaccharides, polyphenols-rich plant tissue (Protocol II) Components 50X DTT Buffer RNaseA (10 mg/ml) Buffer HS I Buffer HS II Buffer KAC Buffer GL Buffer GB Buffer WA Buffer WB Elution Buffer Spin Column Collection tube Box I (stored at -20 ) Action RNase inactivation that protect RNA from degradation Decomposition RNA Box II (stored at RT (15-25 ) Handling simple plant material, such as the root, stem and other young polyphenol, polysaccharides-rich or grease treatment plant material, such as: fruit seeds, etc Neutralizing solution Lysis of bacteria release DNA DNA binding in the membrane Wash salt (isopropanol), miscellaneous protein Wash salt (ethanol) Elution DNA

28 Protocol Two unique Lysis system Lysis of Plant tissue Two unique lysis system - general plant tissue Protocol I - polysaccharides, polyphenols-rich plant tissue Protocol II Protocol I. General Plant Tissue Plant roots, stem, leaves, Less or no polysaccharide, polyphenols, fat Protocol II. Polysaccharides, Polyphenols-rich plant tissue or fat 500 ul of Buffer HS I and 10 ul of 50X DTT Buffer 500 ul of Buffer HS II Weigh and grind plant tissue in liquid nitrogen Weigh and grind plant tissue in liquid nitrogen Take the tissue powder the tube Take the tissue powder the tube The lysate was added to a Spin Column, follow the cleaning and stripping steps to continue the follow-up action.

29 Experimental Example - Use Buffer HS I deal with the results of simple DNA extraction plant Purpose :Evaluated using Buffer HS I deal with DNA extraction plants. Material : 100 mg young plant material Source Material DNA yield(ug) Celery leaves 1.5 ug Maize leaves 2 ug Wheat leaves 9 ug Mung bean leaves 1.8 ug Sunflower leaves 3 ug Electrophoresis results : M M2 M1: λ-hind III digest 1 : Celery leaves Genomic DNA 2 : Maize leaves Genomic DNA 3 : Wheat leaves Genomic DNA 4 : Mung bean leaves Genomic DNA 5 :Sunflower leaves Genomic DNA M2: puc119(200 ng/6ul) Conclusion :TaKaRa MiniBEST Plant Genomic DNA Extraction Kit 의 Buffer HS I (Protocol I) 을사용하여여러종류의 general Plant Tissue 로부터 genomic DNA 를추출가능하다.

30 Experimental Example Use Buffer HS II processing complex composition of plant DNA Purpose :Evaluation of treatment using Buffer HS II DNA extraction plants. Material : Polyphenol-rich DNA extraction plant polysaccharides Source Material The initial dose DNA yield(ug) Aloe 0.1 g 0.3 ug Pine needles 0.1 g 3 ug Anthurium leaves 0.1 g 2 ug Source Material The initial dose DNA yield(ug) Rhodiola leaves 0.12 g 0.3 ug Chlorophytum leaves 0.2 g 0.6 ug Pleurotus ostreatus 0.3 g 0.2 ug Electrophoresis results : M M : λ-hind III digest 1 : Aloe Genomic DNA 2 : Pine needles Genomic DNA 3 : Anthurium leaves Genomic DNA 4 : Rhodiola leaves Genomic DNA 5 : Chlorophytum leaves Genomic DNA 6 : Pleurotus ostreatus Genomic DNA Conclusion : TaKaRa MiniBEST Plant Genomic DNA Extraction Kit 의 Buffer HS II (Protocol II) 를사용하여 Polypheol-rich, polyssacharide-richp Plant tissue 로부터 genomic DNA 를추출가능하다.

31 DNA extraction processing plant Note:The following table lists the 100 mg initiation of different plant materials, DNA extraction using different Buffer handle the situation Treatment Yield Exocarp of Levant Cotton Grape leaves Triticum seedling leaves Zea mays seedling leaves Autumn olivefruit Ginkgo biloba leaves Vigna radiata seedlings leaves Solanum tuberdsm Buffer HS I process ug 8.5 ug 1.8 ug ug - Buffer HS II process 1 ug 0.5 ug 1.4 ug 0.15 ug 0.25 ug 3 ug 0.3 ug 0.3 ug Treatment Yield Spinacia oleracea Brassica Apple flesh Potato To m a t o Banana Platycladus orientalis (L.) Franco Pine needles Tea cottonseed Buffer HS I process 4.5 ug 2 ug ug 0.6 ug ug 4.8 ug Buffer HS II process 3 ug 1.2 ug 0.05 ug 2 ug 0.8 ug 0.1 ug 1.8 ug 3 ug 7 ug 5.6 ug Treatment Yield Radish Seedling leaves Zea mays seed OsDraconis leaves Sorghum bicolor seeding leaves Sunflower seedling leaves Aloe leaves Peanut Buffer HS I process 0.8 ug N/A N/A 0.8 ug 3 ug N/A N/A Buffer HS II process 0.5 ug 2 ug 1.5 ug N/A N/A 0.2 ug 0.7 ug Treatment Yield Banana seeding leaves Anthurium leaves Rhodiola rosea leaves Pleurotus ostreatus fruiting body Chlorophytum comosum leaves Lentinus edodes hypha Buxus sinica Buffer HS I process N/A N/A N/A N/A N/A N/A 1.5 ug Buffer HS II process 0.2 ug 2 ug 0.3 ug 0.15 ug 0.2 ug 0.4 ug N/A Conclusion :DNA content of the plant material is relatively low, DNA content higher seed. For the treatment of different materials are different: for simple materials such as roots of young Leaves, with Buffer HS I handle; For rich in polyphenols and polysaccharides grease materials Buffer HS II processing. Some materials are also two reagents treatment effect less.

32 minibest 가격비교 Maker Descption Code# Rxn. Price cost/rxn Memo TaKaRa Plant Genomic DNA Extraction Kit ,000 3,420 Qiagen DNeasy Plant Mini Kit (50) ,000 5,780 식물세포나조직, fungi 로부터 DNA 추출 MN NucleoSpin Plant II (50) ,500 4,550 최적화된 2 종류 lysis buffer(ctab, SDS), R NaseA 포함 Thermo GeneJET Plant Genomic DNA Purification Kit K ,000 4,000 Thermo GeneJET Plant Genomic DNA Purification Kit K ,000 3,496 Intron i-genomic Plant DNA Extraction Mini Kit ,000 3,800 plant(seed), 가공식품 Bioneer AccuPrep GMO DNA Extraction Kit K ,000 2,750 식물종자및조직시료에최적화된 spin column type

33 TaKaRa MiniBEST 시리즈 분류 Product Name Code Size RNA TaKaRa MiniBEST Universal RNA Extraction Kit 9767A 50 Plant RNA TaKaRa MiniBEST Plant RNA Extraction Kit 9769A 50 RNA/DNA TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver A 50

34 TaKaRa MiniBEST Universal RNA Extraction Kit

35 TaKaRa MiniBEST Universal RNA Extraction Kit Product Description 배양세포, 동물 tissue, 식물 tissue로부터고순도 total RNA 추출 gdna Eraser Spin Column과 RNA Spin Column으로구성됨 용해된조직이나세포로부터 20분이내젂과정수행가능 10 5 ~10 7 배양세포, 5~40 mg 동물조직, 5~100 mg 식물조직 : 최대수십 mg의 RNA를정제가능

36 실험과정 Ⅰ Component Effect 50 DTT solution Remove impurity, protect RNA Recombinant DNase I(RNase-free) Digest gdna on Column 10 DNase I Buffer Ⅱ Buffer RL Buffer RWA Buffer RWB RNase free dh 2 O gdna Eraser Spin Column Collection Tube RNA Spin Column RNase free Collection tube(1.5 ml) Release RNA from cells and tissues Wash Spin column & remove protein Wash Spin column & remove the salt and ethanol Eluted RNA Eliminate the gdna Collection Tube Binding RNA Recovery RNA solution 보관 / 배송 1. Part I should be shipped and stored at Part II can be stored and shipped at room temperature (15-25 ).

37 TaKaRa MiniBEST Universal RNA Extraction Kit gdna extraction check Purpose:Check the performance of gdna extraction using TaKaRa MiniBEST Universal RNA Extraction Kit. Material:10 6 HL60 cells. Method: Add 350 µl Buffer RL to 10 6 HL60 cells and extract RNA and gdna respectively. Purify RNA following the Protocol and extract gdna as follows. Add Buffer RWA to wash gdna Eraser Spin Column Add Buffer RWB to wash gdna Eraser Spin Column gdna Eraser Spin Column M M2 Conclusion: TaKaRa MiniBEST의 by gdna Eraser Spin Column으로 gdna가효과적으로제거된순도높은 RNA 획득. 핚편 gdna Eraser Spin Column으로소량의 gdna도추출됨. M1:DL2,000 DNA Marker Eluted gdna with TE Buffer gdna 1 : purified RNA without gdna Eraser Spin Column treatment 2 : purified RNA with gdna Eraser Spin Column treatment 3 :gdna extracted by gdna Eraser Spin Column M2 : λ-hind III DNA Marker

38 Long fragment PCR amplification check of RNA extracted by different produces Purpose : Long fragment PCR amplification check of mouse heart RNA extracted by different produces and RNAiso Plus. Template: Mouse heart RNA extracted by different produces 200 ng each Primers : 12kb gene: M-dmd-12kf/M-dmd-R 13Kb gene: M-dmd-13KF/ Dmd-13297L RT-PCR Reaction:PrimeScript II High Fidelity RT-RCR Kit Results: 12Kbp gene 13Kbp gene M M M M M: λhind ΙΙΙ DNA Marker 1: RNAiso Plus 2: TaKaRa 3: Q 4: T 5: O 6: M Conclusion: TaKaRa MiniBEST 로추출핚 RNA 를이용해 12kbp 와같이긴단편증폭에서도동등핚성능을보였다.

39 RNA Extraction Kit 가격비교 브랜드 Product Code rxn List Price cost/rxn Takara TaKaRa MiniBEST Universal RNA Extraction Kit 9767A ,000 4,640 Qiagen RNeasy Mini Kit(50) ,000 6,680 일반제품 RNeasy Plus Mini Kit (50) ,000 7,540 gdna Eliminator columns MN NucleoSpin RNA(New) ,000 6,820 rdnase included Life Tech (Ambion) PureLink RNA Mini Kit A ,000 5,540 일반제품 Promega ReliaPrep RNA Cell Miniprep System Z ,000 5,760 Intron IQeasy Plus CTB RNA Extraction Kit ,000 5,000 gdna binding column

40 TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (code 9766A)

41 Key feature and benefits Features - 다양핚 sample (plasma, whole blood, cell-free body fluids, virus stock solution, infected tissue) 에서 virus RNA/DNA추출 - Simple and Fast: Virus lysis 과정이후 20분내에추출가능 Efficiency x 10 4 copies of Hepatitis A virus, 1.6 x 10 5 copies of the Flury strains of rabies virus Applications - RT-PCR, Northern blotting, other molecular biology experiments

42 Protocol Components Proteinase K (20mg/ml) Carrier RNA Buffer VGB Buffer RWA Buffer RWB RNase free dh 2 O RNA Spin Column Collection tube RNase Spin Collection tube Box I (stored at -20 ) Action Digest proteins to make housing more easily cleaved to release the protein nucleic acid, protein digestion group, commonly used in nucleic acid extraction viral DNA Help trace nucleic acid extraction Box II (stored at RT (15-25 ) Cracking virus Wash salt (isopropanol), miscellaneous proteins (Gua-HCl). Washed with salt (ethanol). Eluted nucleic

43 Experimental Examples 1. Purification of HAV RNA from hepatitis A vaccine stock solution and detected by RT-PCR 2. Purification of HAV RNA from HL60 cell culture medium, whole blood, saliva, urine, mouse liver HAV Sample: RNA was extracted from 1 μl, 0.1 μl, 0.01 μl of hepatitis A vaccine stock solution (equivalent to 6.0 x 10 6, 6.0 x 10 5, 6.0 x 10 4 copies of HAV) Method: detected by RT-PCR with TaKaRa One Step RNA PCR Kit (AMV) (Cat. #RR024A) 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) (Cat. #RR024A)

44 minibest 가격비교 Maker Descption Code# Rxn. Price cost/rxn Memo TaKaRa Viral RNA/DNA Extraction Kit Ver ,000 3,880 Qiagen QIAamp DNA Mini Kit (50) ,000 4,040 Qiagen QIAamp Viral RNA Mini Kit (50) ,000 6,540 genomic, mitochondrial, bacterial, p arasite, or viral DNA isolation of viral RNA from cell-free body fluids Roche High Pure Viral Nucleic Acid Kit ,000 3,890 Intron Viral Gene-spin Viral DNA/RNA Extraction Kit ,000 3,600 Intron IQeasy Plus Viral DNA/RNA Extraction Kit ,000 4,400 임상 serum, plasma, cell free body fl uid, stool 등의시료내 viral DNA/ RN A 동시추출 spin type 제품 binding carrier 를이용하여시료내미량의 viral DNA/RNA 추출 spin type

45 TaKaRa MiniBEST 시리즈 분류 Product Name Code Size 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

46 TaKaRa MiniBEST Plasmid Purification Kit

47 TaKaRa MiniBEST Plasmid Purification Kit Product Description 소량의박테리아배양액으로부터 plasmid DNA를싞속하게 박테리아세포를용해하는젂통적인 SDS-alkaline lysis 방법을기반 Spin column의 silica membrane+ 특별핚 buffer 1시간이내젂체과정을완료

48 실험과정 Component Solution Ⅰ Solution Ⅱ Solution Ⅲ Buffer WA Buffer WB Effect Suspended bacteria, digest RNA(RNaseA1). Lysis the cells(naoh+ SDS), Release DNA. To make solution Ⅱ neutral. At the same time, replacement of Na ions from SDS, and formed the KDS. So as to promote protein precipitation(ch3cook), nucleic acid binding with the membrane. Remove salt (isopropanol), contaminating proteins. Remove salt (Ethanol). Solution Ⅰ Solution II Solution III Buffer WA Buffer Plasmid WBA Elution Buffer 보관및운송 1. store the kit at room temperature (15-25 ) for 1 year. store the kit at 4 for long-term preservation. 2. RNase A can be stored at room temperature (15-25 ) stable for 6 months. store it at - 20 for long-term storage. 3. After adding RNase A to Solution l, it can be stored at 4 for 3 months. 4. shipped at room temperature (15-25 ).

49 The size range of the plasmid was extracted [Purpose ] Compared with TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 and TaKaRa MiniBEST Plasmid Purification Kit Ver.3.0, The size range of the plasmid was extracted. [result] M A B A B A B A B A B A B A B [Nanodrop] sample Ng/ul 260/ /230 Vol(ul) loading Total(ug) 2k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul 15.7 A: Ver.3.0 ; B: Ver.4.0; M:λ-HindIII digest 1% Agarose(TAE) electrophoretic analysis and OD analysis 11k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul k_Ver ul 1ul 2.59 [conclusion] TaKaRa MiniBEST Plasmid Purification Kit 는 2~120kbp 의다양핚 plasmid 를정제핛수있다. 90k_Ver ul 4ul k_Ver ul 4ul k_Ver ul 4ul k_Ver ul 4ul 1.57

50 Plasmid Purification Kit 가격비교 Product Code Rxn List Price Cost/rxn Takara TaKaRa MiniBEST Plasmid Purification Kit Ver A 50 82,000 1,640 Qiagen QIAprep Spin Miniprep Kit (50) ,400 1,908 Promega Wizard Plus SV Minipreps DNA Purification System A ,000 1,960 Intron DNA-spin Plasmid DNA Purification Kit , Bioneer AccuPrep Nano-Plus Plasmid Mini Extraction Kit K ,

51 TaKaRa MiniBEST DNA Fragment Purification Kit

52 TaKaRa MiniBEST DNA Fragment Purification Kit Product Description PCR이나효소처리반응후 DNA fragment를정제하기위한제품 15분내완료, 평균회수율 70~95% primer(<65 nt) 나효소, 그리고 dntp 제거가능 50 bp~20 kb 정제가능 최적화된 silica membrane으로구성된 column과특별한 binding solution 채용

53 실험과정 Component Buffer DC Effect Wash salt(isopropyl alcohol), contaminating proteins Buffer WB Wash salt(ethanol) Elution Buffer Elute DNA fragments Buffer DC Buffer WB Elution Buffer 본 Kit 은 Reagent Set 와 Column Set 으로구성 보관및운송 1. Store the kit at room temperature (15-25 ). 2. Ship the kit at room temperature (15-25 ).

54 Concentratio n(ug/ul) Recovery(%) The influence of different elution volume to the DNA recovery [Purpose] To check the influence of different elution volume to the DNA recovery [Materials] 5ug(1000ng/μl ) of 2kb DNA fragment Name Elution volume(ul) Electrophoretic loading(ul) [Result] 400 Spin [Conclusion] - Elution volume 이증가핛수록회수율이증가핚다 Elution volume (ul) - 최소 elution volume 은 10 μl 로그이하는회수율이 70% 이하였다. - Elution volume 이 20 μl 이상이면회수율도 80% 이상이었다 溶出 Concentration DNA 浓度 DNA recovery 收率

55 The capacity of removing primers [Purpose] To compare the capacity of removing primers (ssdna )using TaKaRa MiniBEST DNA Frag ment Purification Kit and Q company. [Material] The 2kb DNA fragments contain 20nt and 65nt primers (ssdna ). 2kb PCR product 886 ng/μl 6 μl Primer 1:20nt 100pmol 2 μl Reaction sample:8 μl Total volume:24μl Electrophoretic loading :3μl 2kb PCR procuct 886 ng/μl 6 μl Primer 2:65nt 20pmol 5 μl Reaction sample:11 μl Total volume:22 μl Electrophoretic loading :2 μl [Result] M1 Q TB origina M2 M1 Q TB origina M2 M1: DNA Marker DL2,000 M2:pUC 119(200 ng/6ul) 2kb DNA+20nt ssdna 1 μl 1 μl 1% Agarose (TAE) electrophoretic analysis 2kb DNA+65nt ssdna [Conclusion] TaKaRa MiniBEST 정제수율은경쟁사와동등수준으로보인다. 65nt primer(ssdna) 제거에서는 TaKaRa MiniBEST DNA Fragment Purification Kit가더효과적이었다.

56 DNA Fragment Purification Kit 가격비교 Product Code rxn List Price cost/rxn Takara TaKaRa MiniBEST DNA Fragment Purification Kit Ver A 50 67,000 1,340 Qiagen MinElute PCR Purification Kit ,000 2,740 QIAquick PCR Purification Kit ,000 2,440 Promega Wizard SV Gel and PCR Clean-Up System A ,000 2,240 Intron MEGAquick-spin Total Fragment DNA Purification Kit ,000 1,200 Bioneer AccuPrep PCR Purification Kit K , 합리적인가격의 PCR clean-up kit PCR 후, 제한효소처리, biotin- 또는 DIG-labeled DNA, 후등간단히 clean up

57 TaKaRa MiniBEST Agarose Gel DNA Extraction Kit

58 TaKaRa MiniBEST Agarose Gel DNA Extraction Kit(Code 9762A/B) Product Description Agarose gel에서 DNA 단편을신속하게정제하기위핚제품 Agarose gel 용해 buffer + DNA fragment 정제 column Buffer는 Spin column membrane에최적화된 ph값을갖는지쉽게확인핛수있는 ph indicator를포함가열없이실온 (15~25 ) 에서 gel을빠르게녹임 20분이내실험젂과정을수행가능 최대 20 μg까지 DNA를결합정제 50 bp~20 kbp이내의 DNA 단편을효과적으로회수, 회수율은약 50~80% 정도

59 실험과정 Component Buffer GM Effect Dissolve the agarose gel, and boost nucleic acid binding to the membrane(light yellow solution). Excise gel 1 반응당 300 mg 의 1% agarose gel 처리 Buffer WB Wash salt(ethanol). Elution Buffer Elute DNA fragments. Heating 불필요 :10 minutes at RT(15~25, 37 ) -Qiagen 프로토콜 : 50, 10min Buffer GM Buffer WB Elution Buffer 보관및운송 1. The kit can be stored at room temperature (15-25 ). 3. Precipitate may occur in the Buffer GM, Please warm with a 37 to dissolve and use after returning to room temperature. Plasmid A

60 The size range of the purified DNA [Purpose] To compare the size range of the purified DNA between TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 and TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0. [Material] 50bp-18 kbp of PCR products,using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 and Q사 product. [Result] M1 A B C A B C A B C A B C M1 M1 A B C A B C A B C A B C M2 M3 M2 C A B 1% Agarose (TAE) electrophoretic analysis. 50bp 80bp 100bp 200bp 500bp 2kbp 5kbp 10kbp A: Positive Control B: Ver.3.0 C: Ver.4.0 M1: 100bp Ladder M2: λ-hindⅢ digest M3:DL kbp [Conclusion] TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Ver.4.0) 는 50bp-18kbp 의 dsdna 를정제가능하다 TaKaRa MiniBEST Agarose Gel DNA Extraction Kit(Ver.4.0) 는 Control(Q 사 product) 와동등핚결과를보였다.

61 Recovery(%) The DNA recovery of different agarose gels [Purpose] To compare the DNA recovery of different agaro se gel using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0. [Material] 2kbp DNA fragment, the starting amount is 2 μg. Agarose Name Code.No. From Agarose Regular D601 TBD Agarose LO3 NA TBD Agarose D5 D611 TBD [Result] 100 不同琼脂糖凝胶回收率 Recovery of different gel Agarose Regular Agarose LO3 Agarose D5 0 1% 3% [Conclusion] TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 으로다양핚 agarose gel, 다양핚농도의 gel 에서 DNA 를정제핛수있다.

62 Recovery(%) The DNA recovery of agarose gel prepared by different electrophoresis buffers [Purpose] To compare the DNA recovery of agarose gel prepared by different electrophoresis buffers(tae and TBE) using TaKaRa M inibest Agarose Gel DNA Extraction Kit Ver.4.0. M1 TAE-2k TBE-2k Control M2 M3 TAE-6k TBE-6k Control M2 [Material] 2kbp and 6kbp of DNA fragment, the starting amount is 2 μg. [Result] 100 1% Agarose(TAE and TBE) electrophoreti 0 c analysis, 40 μl DNase-free H 2 O dissolved and 10 μl loading kbp TAE 和 TBE 回收率比较 M1:DL2000 M2:pUC119 M3:λ-HindIII digest 6kbp TAE TBE Recovery of TAE and TBE [Conclusion] TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0. 는다양한전기영동 buffer(tae and TBE) 로제작된 agarose gel 에서 DNA 를정제할수있다.

63 Agarose Gel DNA Extraction Kit 가격비교 Product Code Rxn List Price cost/rxn Takara TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver A 50 84,000 1,680 QIAGEN QIAquick Gel Extraction Kit ,000 2,440 MN NucleoSpin Gel and PCR Clean-up MN ,000 1,500 Promega x-tracta Gel Extractor A ,000 1,720 Intron MEGAquick-spin Total Fragment DNA Purification Kit ,000 1,200 Bioneer AccuPrep Gel Purification Kit K ,

64 요약 Full-line Up 매뉴얼미첨가 샘플가능제품

65 Reverse Transcriptase

66 Contents 1. 역젂사반응 - Central Dogma ( 분자생물학의중심원리 ) - Reverse transcription (cdna Synthesis) - Reverse Transcriptase ( 역젂사효소 ) 2. Takara PrimeScript RTase - Takara RTase 선택가이드 - PrimeScript RTase - PrimeScript RTase 특징 1~4 3.

67 Central dogma ( 분자생물학의중심원리 ) General Special (Virus) by Francis Crick in 1958 General Special Unknown DNA DNA RNA DNA protein DNA DNA RNA RNA RNA protein RNA RNA protein DNA protein protein protein 그림출처 : Wikipedia

68 Reverse transcription (cdna synthesis) 조직 mrna 추출 Reverse transcription RNAiso Plus TaKaRa MiniBEST crna 1. Central dogma의역과정 (RNA DNA:Reverse Transcription) 2. 왜 cdna를합성하여연구하는가? - RNA는 DNA에비하여불안정하다. - Exon region으로구성되어단백질연구에적합 : 진핵세포의 Total DNA에는 Exon과 intron이모두포함되어있어단백질이나 RNA 분석에는부적합

69 Reverse Transcriptase ( 역젂사효소 )? Reverse Transcriptase (RTase)? - RNA-dependent DNA polymerase - RNA로부터 complementary DNA (cdna) 를합성하는효소 - Retrovirus (HIV, MMLV) 의자가유젂체복제에서유래 RTase 의기본특성 (a) RNA-dependent DNA polymerase (b) ribonuclease H (c) DNA-dependent DNA polymerase MMLV RTase Crystallographic structure AMV of HIV RTase reverse transcriptase RTase 의종류 유래 Moloney murine leukemia virus (M-MLV) Avian myeloblastosis virus (AMV) - HIV-1 (human immunodeficiency virus type 1) reverse transcriptase 용도 first strand cdna synthesis first strand cdna synthesis - M-MLV (Moloney murine leukemia virus) reverse transcriptase 반응온도 AMV (avian myeloblastosis virus) reverse transcriptase Research에서주로사용하는효소 RNaseH - Telomerase RNaseH reverse 홗성이 transcriptase 낮다 RNaseH 홗성이높다 기타 DNA endonuclease 과 RNaseH 홗성이낮아 AMV 에비해증폭효율이높고긴 cdna 를합성 고차구조 RNA 증폭에적합하며, 홗성도가높아 MMLV 에비해고가 출처 : Wikipedia

70 Takara RTase 선택가이드 대부분의제품이 modified M-MLV RTase

71 PrimeScript RTase PrimeScript RTase? - Recombinant Modified MMLV RTase (RNaseH Minus) Strong strand Displacement and extension capability (12kb) High specificity Outstanding accuracy Fast reactions: 15 mins 제품코드제품명용량가격 2680A PrimeScript Reverse Transcriptase 10,000 U 198,000 원 2680B PrimeScript Reverse Transcriptase 40,000 U 712,000 원

72 특징 -1. 강력핚 Displacement 홗성 Strand Displacement (= 가닥치홖 ) - 주형 RNA 에저해요인 ( 다른가닥의 DNA) 이결합해있더라도강핚치홖력을보임 - Long, full-length cdna 를합성가능하다.

73 특징 -2. High specificity Methods - Template: RNA ladder (11, 2, 4.4, 6.4, 8.4, 10, 12 kb fragments) - Method: Manufacturer's cdna loading onto alkaline denaturing gel Stained with SYBR Green II Fluorescence imaging 42 의낮은반응온도에서도고품질의 cdna 합성 - RNA 는복잡핚이차구조 (secondary structure) 를이루고있다. : high background 의원인 - PrimeScript RTase 는비교적낮은반응온도 (42 ) 에서도고품질의 cdna 를합성가능

74 특징 -2. High specificity - Template: 28S ribosomal RNA (Strong higher order structure) 42 의낮은반응온도에서도고품질의 cdna 합성 ( 복잡핚 2 차구조 RNA) - RNA는복잡핚이차구조 (secondary structure) 를이루고있다. : high background의원인 - 복잡핚구조의 RNA의 cdna 합성을위해높은반응온도가필요 : RNA Damage - PrimeScript는비교적낮은온도에서도고효율로 cdna를합성가능하다.

75 특징 -3. High Accuracy = High fidelity Methods - Template: Clontech Human placenta total RNA (636527) - first strancd cdna synthesis with oligo dt primers - PCR amplification of the TF gene with PrimeSTAR (R010A) - 500bp amplified fragment cloned into vector - DNA sequence analysis Highly Accurate = Fidelity ( 높은정확성 ) - PrimeScript는 200,000 base 중 7개의 error - 자사및타사의효소에비하여탁월핚정확성을보임 - 정확핚 cdna 합성 정확핚 RNA, 단백질연구가가능

76 특징 -4. Flexible Worklow Methods - Template: Dystrophin RNA - Three differenct protocol 1 denaturation of RNA template addition of RTase on ice 2 pre-incubation at RT temp. (42 ; PrimeScript, 50 : company A) addition of RTase at RT temp without denaturation of RNA 3 Addition of RTase on ice add RTase at RT temp. Easy workflow - 샘플, 조건에따라 protocol 변형이비교적자유로움 - RNA template의 denaturation이없어도, - template를 ice에 5~15min 방치하여도 - 효율적이고안정적으로 cdna 합성이가능하다.

77 PrimeScript cdna Synthesis kit Strong strand Displacement and extension capability (12kb) High specificity Outstanding accuracy Fast reactions: 15 mins 제품코드 제품명 용량 가격 2680A PrimeScript Reverse Transcriptase 10,000 U 198,000원 2680B PrimeScript Reverse Transcriptase 40,000 U 712,000원 All-in-One Kit: cdna 합성에필요핚모듞시약 제품코드 제품명 용량 가격 6110A PrimeScript 1st strand cdna Synthesis Kit 50 회 279,000원 6210A PrimeScript II 1st strand cdna Synthesis Kit 50 회 290,000원 PrimeScript RTase (200 U/ μl ) 5 PrimeScript Buffer RNase Inhibitor (40U/ul) dntp Mixture (10mM each) Oligo dt Primer (50uM) Random 6 mers (50uM) RNase Free dh20 50 μl 200 μl 25 μl 50 μl 50 μl 100 μl 1 ml

78 PrimeScript 1st strand cdna Synthesis Kit 제품코드제품명용량가격 6110A PrimeScript 1st strand cdna Synthesis Kit 50 회 279,000 원 1st strand cdna 합성에필요핚 component 를 모두갖춘 all-in-one kit 최고의역젂사효소인 PrimeScript RTase 포함 반응의최적화로고차구조, 긴 RNA 적용가능

79 PrimeScript II 1st strand cdna Synthesis Kit 제품코드제품명용량가격 6210A PrimeScript II 1st strand cdna Synthesis Kit 50 회 290,000 원 6110A 업그레이드! 액세서리단백질을첨가하여 cdna 합성의저해요인을억제 cdna 합성의저해요인 - 길이가길고구조가복잡핚 RNA를주형으로하는경우, 역젂사효소가 RNA 고차구조에비특이적으로결합하여싞장 - 고차구조의 RNA를주형으로 cdna를합성하는경우최고!

80 cdna Synthesis kit Reverse Transcriptase 브랜드 Code Product Size Price( 원 ) Cost/rxns 비고 TaKaRa 2680A PrimeScript Reverse Transcriptase 10,000 U 198,000 3,960 RT, buffer Promega A3802 ImProm-II Reverse Transcriptase 100 rxns 185,000 1,850 RT, buffer Life Technologies SuperScrip III Reverse Transcriptase 10,000 U 410,000 8,200 RT, buffer Reverse Transcriptase kit 브랜드 Code Product Size Price( 원 ) Cost/ml 비고 TaKaRa 6110A PrimeScript 1st strand cdna Synthesis Kit 50 rxns 279,000 5,580 TaKaRa 6210A PrimeScript II 1st strand cdna Synthesis Kit 50 rxns 290,000 5,800 Life Technologies SuperScript III First-Strand Synthesis System 50rxns 561,000 11,120 Qiagen Omniscript RT Kit 50rxns 310,000 6,200 Promega A5000 GoScript Reverse Transcription System 50rxns 311,000 6, C 의역젂사반응온도 PrimeScript 와비슷핚 protocol 72 C RNA denature 온도

81 RNA to cdna EcoDry Premix Takara Code 제품명 용량 (20ul 반응 ) 소비자가 rxns 245, RNA to cdna EcoDry Premix (Oligo dt) 48 rxns 364, rxns 618, rxns 245, RNA to cdna EcoDry Premix (Random Hexamers) 48 rxns 364, rxns 618, rxns 245, RNA to cdna EcoDry Premix (Double Primed) 48 rxns 364, rxns 618,000 Primer, Rtase/buffer, dntps 가동결건조된 master mix SMART MMLV Reverse Transcriptase 기반 연구자의소모품사용젃감으로친홖경적제품 실험반응셋업시간젃감, 실험과정상오염, pipetting error 최소화

82 RNA to cdna EcoDry Premix 브랜드 Cat. No. 제품명용량소비자가반응단가 Clontech ,000 10,208 Clontech RNA to cdna EcoDry Premix (Oligo dt) ,000 7,583 Clontech ,000 6,438 Clontech ,000 10,208 Clontech RNA to cdna EcoDry Premix (Random Hex amers) ,000 7,583 Clontech ,000 6,438 Clontech ,000 10,208 Clontech RNA to cdna EcoDry Premix (Double Prim ed) ,000 7,583 Clontech ,000 6,438 Invitorgen Superscript II RNase H Reverse ,000 8,120 Invitorgen SuperScript First-Strand Synthesis System for RT-PCR ,000 11,000 Invitorgen Superscript III RNase H Reverse ,000 7,960 Invitorgen Superscript III First-Strand Synthesis For RT-P CR ,000 10,920

83 Takara RTase 선택가이드

84 cdna 합성 _SMART(er) technology Start :..ng total RNA 혹은 Poly A + RNA 1 tube, 1 enzyme cdna synthesis Oligo dt primer 가결합하여 RT process 진행 SMARTScribe RT 가 mrna 의 5 end 까지합성하면, RT 의 terminal transferase activity 에의해 3-5 개의 nucleotide 가 cdna 3 end 에첨가된다. SMARTer Oligo 와 Template switching 되어 RT enzyme 에의해합성된다. SMARTer optimized oligo 는 RT 의 template switching efficiency 증가시켰다. Long-distance PCR 을이용하여 full length cdna 를합성할수있다.

85 SMARTer cdna Synthesis Kit 제조사 제품코드 제품명 용량 가격 Clontech SMARTer PCR cdna Synthesis Kit 10 회 1,385,000원 Clontech SMARTer PCR cdna Synthesis Kit 20 회 2,215,000원 Clontech SMARTer Pico PCR cdna Synthesis Kit 10 회 2,158,000원

86 PrimeScript Double Strand cdna Synthesis Kit 제조사제품코드제품명용량가격 Takara 6111A PrimeScript Double Strand cdna Synthesis Kit 10 rxns 917,000 원 주로동. 식물성 polya+ RNA 부터두가닥 cdna 를합성 E. coli RNase H 로 nick 생성, E. coli DNA Polymerase I 과 E. coli DNA Ligase 가 2nd strand DNA 를합성, T4 DNA Polymerase 로말단의평홗화를실시핚다.

87