basic protocol - wako-chem.co.jp protocol マークのつ ... rna extraction (isogen,...

Download Basic Protocol - wako-chem.co.jp  Protocol マークのつ ... RNA Extraction (ISOGEN, ISOGEN-LS) 454 RNA Extraction (ISOGENⅡ) 457 Immuno-AptamerTM

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  • 25Basic P

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    Basic Protocol

    PCR 438PhosphorylationT4 Polynucleotide Kinase 441 442LigationLigation Pack 444Transformation/Transduction 446Agarose for Electrophoresis 449RNA Extraction ISOGEN, ISOGEN-LS 454RNA Extraction ISOGEN 457Immuno-AptamerTM North-Western 460DNA Extractor WB Kit 462microRNA Isolation Kit, Human Ago2, microRNA Isolation Kit, Mouse Ago2 465microRNA Cloning Kit Wako 470

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    PCR

    1. PCR PCR Polymerase Chain Reaction

    DNADNAPCRPCR DNA

    DNADNA3 n

    DNA 2n 19496DNA255 60DNA 372 741DNA 2 nDNA 2n 20 100 100

    2. PCR PCRDNA

    Taq DNA PCR

    Taq DNA 1 2.5 units/100 l

    dNTP dNTP 20 200 mol/l

    dNTPdNTP100 l 20 mol/l dNTP 2.6 g DNAMg2 dNTP0.5 2.5 mmol/l

    DNA EDTA

    18 28GC

    50 60%Tm 55 802 0.1 0.5 mol/l

    Taq

    DNA 94 96 15 30

    PCRDNA

    Tm 5 55 600.2 mol/l 72

    1 35 100

    40

    pH PCR 10 50 mmol/l Tris-

    HCIpH 8.3 8.8 at 20PCR

    3. PCR

    Tm 3 3 GC50 60

    3GC

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    Gene TaqGene Taq

    MMarker 6/Sty digest

    DNALane 1 ColE1 DNA 500 bpLane 2 DNA 5 kbpLane 3 DNA 15 kbp

    0.8% Agarose S100 V30

    DNA 1 g10 Gene Taq Universal Buff er 5 ldNTP Mixture2.5 mmol/l each 4 lprimer-forward20 pmol/l 1 lprimer-reverse20 pmol/l 1 lGene Taq Gene Taq NT5 units/l 0.5 lddH2O up to 50 l

    ColE1 DNA 500 bp DNA5 kbp15 kbp

    94 30 55 30 2072 1

    10 l

    941

    94306812

    30

    725

    10 l

    PCR GeneTaq GeneTaq NT

    PCRDNA

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    4. T4 gene 32 protein PCR

    T4 gene 32 protein T4 gene 32 protein T4 DNA

    T4 gene 32 protein DNADNA site-specifi c mutagenesis DNA PCR

    T4 gene 32 protein PCR 0.1 ng DNApBR322

    T4 gene 32 protein PCR

    DNApBR322 0.1 nghemin 10 0.001 g/l10 Gene Taq Universal Buff er 5 ldNTP Mixture2.5 mmol/l each 4 lprimer-forward20 pmol/l 1 lprimer-reverse20 pmol/l 1 lGene Taq5 units/l 0.5 lT4 gene 32 protein 0 2 g 40 ng/lddH2O up to 50 l

    94305530 25 721

    10 l

    2 Agarose S100 V30MMarker 5X174/Hinc

    1 Kreader, C. A. : Appl. Environ. Microbiol., 62, 110219962 Bittner, M., Burke, R. L. and Alberts, B. M. : J. Biol. Chem., 254, 956519793 Schwarz, K., Hansen-Hagge, T. and Bartram, C. Nucl. Acids Res., 18, 107919904 Panaccio, M. and Lew, A. : Nucl. Acids Res., 19, 115119915 Kaspar, P., Zadrazil, S. and Fabry, M. : Nucl. Acids Res., 17, 361619896 Huang, H. and Keohavong, P. : DNA AND CELL BIOLOGY, 15, 58919967 , : , 7, PCR, p.1591997

    1g/l PCR 40 ng/l T4 gene 32 proteingp321g/l

    p.152

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    4 DNA

    5DNA 5 1 50 pmol 2

    5 Kinase Buff er C 10 l[-32P]ATP111 TBq/mmol. 0.37 MBq/l 50 pmol15 lT4 Polynucleotide Kinase 20 units ddH2O 50 l 3730

    10Kinase Buff er A : 0.5 mol/l Tris-HCIpH 7.6, 0.1 mol/l MgCl2, 50 mmol/l DTT, 1 mmol/l Spermidine, 1 mmol/l EDTA5Kinase Buff er B : 250 mmol/l lmidazole-HCIpH 6.4,

    90 mmol/l MgCl2, 25 mmol/l DTT, 30w/vPEG60005Kinase Buff er C 250 mmol/l lmidazole-HCIpH 6.4,

    90 mmol/l MgCl2, 25 mmol/l DTT, 0.5 mmol/l Spermidine, 0.5 mmol/l EDTA, 0.5 mmol/l ADP, 5 nmol/l ATP, 24w/vPEG6000

    PhosphorylationT4 Polynucleotide Kinase DNA RNA 5OH

    5 3

    1DNA1 5 1 50 pmol 2

    10 Kinase Buff er A 5 l [-32P]ATP111 TBq/mmol. 0.37 MBq/l 50 pmol15 lT4 Polynucleotide Kinase 10 20 units ddH2O 50 l 3730

    2DNA1 5 1 50 pmol 2

    5 Kinase Buff er B 8 l [-32P]ATP111 TBq/mmol. 0.37 MBq/l 50 pmol15 lT4 Polynucleotide Kinase 20 units ddH2O 40 l 3730

    1 2

    PCR PCRPCRPCR

    3

    DNA5pmol of 5ends g of DNA/bp of DNA660 g/mol/bp1012 pmol/mol2 ends/molecule 20 mer DNA1 g 150 pmol of 5end 150 pmol of 5end0.000001/20 3301,000,000,000,0001 1 kbp DNA1 g 1.52 pmol 3 pmol of 5end 3 pmol of 5end0.000001/1,000 6601,000,000,000,0002

    1 DNASepharose CL-4B T4 Polynucleotide Kinase DNADNA

    2 1 pmol5DNA1003.3102 g1,000 3.3 101 g

    3 1/5 1/10

    1 5 150 pmol10 Kinase Buff er A 5 l 10 mM rATP 5 l 1 mM T4 Polynucleotide Kinase 20 unitsddH2O up to 50 l 37 30

    7010//

    2PCR 5 150 pmol5 Kinase Buff er B 8 l 10 mM rATP 5 l 1 mM T4 Polynucleotide Kinase 20 unitsddH2O up to 40 l 37 30

    //

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    2 Alkaline Phosphatase

    3 DNA

    4 BAP 100CIP/

    5

    Cloning Vector pTS1 DNA, Hinc TreatedCode No. 301-10131

    2 PCR Taq PCR DNA

    3 1 AT4 DNA Polymerase 3 5Exonuclease 5 3Polymearse 5 3Blunting-Convenience Kitp.131 T4 DNA Polymerase

    dNTPs 10 Blunting Buff er

    PCR0.1 10 pmol 17 l 6

    10 Blunting Buff er 2 lT4 DNA Polymerase 1 lTotal 20 l 37 5

    //20

    6 DNADNA0.110 pmol17 lpUC19 DNA2,686 bp1 g 1 pmol of 5end

    1 pmol of 5end 0.000001 g/2,686 bp 660 1012 2

    PCR1

    DNA 5PCR 5PCR

    PCRDNA 5OHPCRDNA 5OHT4 Polynucleotide Kinase

    Phosphorylationp.441

    1.

    DNATaq DNA Polymerase PCRDNA 3 1 AT TA PCRDNADNA

    DNA

    2. 3 A PCR

    1 1 10 g

    DNA1//TEpH 8.0 100 g/mlDNA1 DNA DNA

    5

    1DNA

    Alkaline Phosphatase 2

    DNA 10 g 44 l 3

    10 AP Buff er 5 lAlkaline Phosphatase 1 2 lTotal 50 l 50 30 60

    50 l 4

    ddH2O 49 l0.5 M EDTApH 8.0 1 l 5 mM 65 30 75 10

    / /TEpH 8.0 100 g/mlDNA5

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    3 Blunting-Convenience Kit Ligation-Convenience Kit

    2 Ligation Mix 2 Ligation Mix T4 DNA LigaseATPDTT

    DNADNA

    DNA 200 bp 600 bp 1,000 bp 3,000 bp 1 1 1 1 5 5 210 0.52

    SmapUC19 DNA0.03 pmolSmaDNA0.015 pmol, 0.03 pmol, 0.06 pmol, 0.15 pmol, 0.3 pmol165

    DNApmol g bp 1,515g pmol bp 0.00066

    pUC19 DNA 2,686 bp 0.03 pmol DNA 53 ng

    DNADNAddH2O to 10 l2 Ligation Mix 10 lTotal 20 l 16 5 30

    4 2 Ligation Mix

    10 10DNA ECOS Competent E.coli

    p.158ECOS Competent E.coli 5

    5 PCR M13pUC19 DNA

    Gene RED PCR Mixp.62 2M13 Primer MixCode No. 312-07651PCR

    Gene RED PCR Mix 1.5 ml2M13 Primer Mix50 lPCR

    Gene RED PCR Mix 25 l2 M13 Primer Mix 25 lTotal 50 l

    PCR94 3 94 20 55 20 25 cycles72 X 7

    PCR 5 10 l8

    7 X DNA 1 kbp 101 kbp 10

    8 Gene RED PCR Mix Loading Dye Glycerol PCR

    M13no insert PCRpUC18/19 121 bp pGEM-T easy 250 bp pT4Blue2 388 bp

    pBluescript 247 bp pT7Blue 172 bp ZAP 244 bp

    1 Michael R. Green and Joseph Sambrook : Molecular

    Cloning, A Laboratory Manual, 4th ed.2012

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    LigationLigation Pack

    1. Ligation Pack 1 10 Ligation Buff er 1 ml 500 mmol/l Tris-HCIpH 7.9 100 mmol/l MgCl2 200 mmol/l DTT 10 mmol/l ATP2 2 mg/ml BSA Solution1 ml3 20 mmol/l Spermidine100 l4 20 mmol/l Hexamine Cobalt ChlorideHCC100 l5 H2O1 ml 2 6 Enzyme SolutionT4 DNA Ligase300 units/l100 l

    2.

    DNARevie, D2

    3. Ligation Pack 150 l Enzyme SolutionT4 DNA

    Ligase 1 l Enzyme Solution Enzyme Solution Ligation Pack

    12 16 30 16 37

    4 T4 DNA Ligase 5 3

    16 26 10 100 Ligation PackEnzyme Solution1 l

    1

    T4 DNA

    T4 DNA Ligase NaCl 150 mmol/l

    DNA

    BSA 50 250 g/ml

    4. Ligation Pack

    2. 3. Ligation Pack DNA

    DNA DNApUC, pBR

    TE DNA TE

    aDNA 0.05 0.5 gDNA10 Ligation Buff er 2 l2 mg/ml BSA Solution 2.5 lEnzyme Solution 0.5 1 lddH2O up to 20 l16 30 16

    DNA 1.5 kbp

    DNA Ligation transformation

    DNApUC19/EcoR DNA1.5 kbp

    DNA 50 ngDNA1.5 kbpvector 0.1,0.3,1,3,10,20,5010 Ligation Buff er 2 l2 mg/ml BSA Solution 2.5 lEnzyme Solution 0.5 lddH2O up to 20 l16 30 16

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    DNA 50 ng DNA DNA 10 Ligation 30 Ligation Ligation 30

    bDNA 0.05 0.5 gDNA10 Ligation Buff er 2 l2 mg/ml BSA Solution 2.5 l20 mmol/l Spermidine 1 lEnzyme Solution 1 2 lddH2O up to 20 l16 30 16

    DNA DNA

    DNADNA 10 2010 20 l

    DNADNA10 Ligation Buff er 2 l2 mg/ml BSA Solution 1 l20 mmol/l Spermidine 1 l20 mmol/l Hexamine Cobalt Chloride 1 lEnzyme Solution 1 2 lddH2O up to 20 l16 30 16

    ATP ATP

    DNAPyPuN

    DNADNA

    PCRPCRDNA

    T4 DNA Ligase

    PEGPEG

    1 Rusche, J. R. et al. Nucl. Acids Res., 13, 199719852 Revie, D. et al. Nucl. Acids Res., 16, 1030119883 Berger, S. L. and Kimmel, A. R. Methods in Enzymol., 152, 34319874 Sambrook

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