Type-it Mutation Detect PCR プロトコールとトラブルシューティング ( /2008)

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1 June 2008 Type-it Mutation Detect PCR PCR SNPs 2 3 QIAxcel Agilent 2100 Bioanalyzer Sample & Assay Technologies

2 MSDS material safety data sheet Operon Biotechnologies TE 100 µm 20 2 Type-it Mutation Detect PCR 06/2008

3 Type-it Mutation Detect PCR Kit HPLC TE10 mm Tris 1 mm EDTA ph µm4 3 PCR PCR PCR ABI PRISM 3100 Genetic Analyzer Applied Biosystems xl Genetic Analyzer Applied Biosystems xl DNA Analyzer PCR PCR HPLC Handbook Appendix C Appendix D PCR PCR PCR 3 4 TE 20 Type-it Mutation Detect PCR 06/2008 3

4 3. 10x 2 µm * 50 µm 50 pmol/µl 100 µm 100 pmol/µl 20 µl 10 µl TE 500 µl 500 µl * µm µm 10x Type-it Mutation Detect PCR QIAxcel Agilent 2100 Bioanalyzer 3 5 bp 1 PCR PCR PCR Type-it Mutation Detect PCR Kit Type-it Mutation Detect PCR 06/2008

5 4. PCR PCR >200 bp 2,000 bp 1.3 > bp 1,000 bp > bp 750 bp bp 500 bp <20 bp* 250 bp * 20 bp PCR 20 bp MetaPhor agarose FMC Bioproducts bp 5. QIAxcelPCR QIAxcel QX DNA High 15 bp 5 kb bp Resolution Cartridge 3 5 bp 500 bp 1 kb 50 bp 1 5 kb bp QX DNA Screening PCR 15 bp 5 kb bp Cartridge bp 500 bp 1 kb bp 1 5 kb 500 bp Type-it Mutation Detect PCR Kit PCR QX DNA High Resolution Cartridge 500 bp 1.5 kb Type-it Mutation Detect PCR 06/2008 5

6 6. PCR Agilent 2100 Bioanalyzer DNA LabChip Kit ,000 bp bp ,000 bp ,500 bp 100 1,500 bp * 500 bp PCR Type-it Mutation Detect PCR Kit PCR ABI PRISM Genetic Analyzer Applied Biosystems xl Genetic Analyzer Applied Biosystems xl DNA Analyzer Beckmann CEQ 8000 CEQ 8800 Genetic Analysis Systems Handbook 45 Appendix D DNA PCR PCR EDTA QIAGEN PCR DNA QIAamp DNeasy DNA REPLI-g Kits QIAamp DNeasy REPLI-g Kit PAXgene Blood DNA System 6 Type-it Mutation Detect PCR 06/2008

7 PCR DNA Handbook 43 Appendix C 2 QIAxcel Agilent 2100 Bioanalyzer 8 PCR QIAxcel Agilent 2100 Bioanalyzer SNaPshot Technology SNPs 13 PCR 1 bp Type-it Mutation Detect PCR 06/2008 7

8 QIAxcel Agilent 2100 Bioanalyzer SNaPshot Technology SNPs PCR µm T m 68 PCR Handbook 38 Appendix B x Q-Solution Handbook 38 Appendix A Q-Solution CoralLoad Dye PCR DNA CoralLoad Dye PCR 95 5 HotStarTaq Plus DNA Polymerase x Type-it Multiplex PCR Master Mix 20 DNA RNAse CoralLoad Dye Q-Solution 8 Type-it Mutation Detect PCR 06/2008

9 2. 7 Q-Solution 8 DNA PCR µl 7 8 Type-it Multiplex PCR Master Mix 1 1 2x Type-it Multiplex PCR Master Mix 3 mm Mg Q-Solution 2x Type-it Multiplex PCR Master Mix 2x Type-it Multiplex PCR 12.5 µl 1x Master Mix* 10x 2.5 µl 0.2 µm 2 µm µl 1x CoralLoad Dye CoralLoad Dye 10x RNase DNA DNA 300 ng DNA ng DNA 25 µl *MgCl 2 3 mm 0.2 µm µm 25 µl 2x Type-it Multiplex PCR Master Mix 1 1 CoralLoad Type-it Mutation Detect PCR 06/2008 9

10 8. Q-Solution 2x Type-it Multiplex PCR Master Mix 2x Type-it Multiplex PCR 12.5 µl 1x Master Mix* 10x 2.5 µl 0.2 µm 2 µm µl 1x CoralLoad Dye CoralLoad Dye 10x Q-Solution, 5x 2.5 µl 0.5x Q-Solution RNase DNA DNA 300 ng DNA ng DNA 25 µl *MgCl 2 3 mm 0.2 µm µm 25 µl 2x Type-it Multiplex PCR Master Mix 1 1 CoralLoad 3. PCR PCR 4. PCR DNA 300 ng PCR 11 9 PCR 95 5 HotStarTaq Plus DNA Polymerase Type-it Mutation Detect PCR 06/2008

11 9. 2x Type-it Multiplex PCR Master Mix PCR Q-Solution 5 95 HotStarTaq Plus DNA Polymerase PCR 60 T m * kb 0.5 kb 0.5 kb DNA * T m = 2 x number of [A+T] + 4 x number of [G+C] Type-it Mutation Detect PCR 06/

12 10. PCRDNA ng * * Handbook 44 Appendix C Table QIAxcel Agilent 2100 Bioanalyzer PCR 12 Type-it Mutation Detect PCR 06/2008

13 PCR µm T m 68 PCR Handbook 38 Appendix B 4 310x Q-Solution Handbook 38 Appendix A Q-Solution CoralLoad Dye PCR 95 5 HotStarTaq Plus DNA Polymerase x Type-it Multiplex PCR Master Mix 20 DNA Q-Solution DNA PCR µl 11 Type-it Multiplex PCR Master Mix 1 1 2x Type-it Multiplex PCR Master Mix 3 mm Mg 2+ Type-it Mutation Detect PCR 06/

14 11. 2x Type-it Multiplex PCR Master Mix 2x Type-it Multiplex PCR 12.5 µl 1x Master Mix* 10x 2.5 µl 0.2 µm 2 µm 3 Q-Solution 5x 2.5 µl 0.5x PCR CoralLoad Dye RNase DNA DNA 200 ng DNA ng DNA 25 µl *MgCl 2 3 mm 0.2 µm µm 25 µl 2x Type-it Multiplex PCR Master Mix PCR PCR 4. PCR DNA 200 ng PCR PCR 95 5 HotStarTaq Plus DNA Polymerase Type-it Mutation Detect PCR 06/2008

15 12. PCR 5 95 HotStarTaq Plus DNA Polymerase PCR 60 T m * kb 0.5 kb 0.5 kb DNA * T m = 2 x number of [A+T] + 4 x number of [G+C] 0.5 kb 0.5 kb 30 Type-it Mutation Detect PCR 06/

16 13. PCRDNA ng * * Handbook 44 Appendix C Table PCR 6 Handbook 45Appendix D PCR 16 Type-it Mutation Detect PCR 06/2008

17 PCR a HotStarTaq Plus DNA Polymerase b c d e PCR 6 HotStarTaq Plus DNA Polymerase PCR DNA 0.2 µm 0.1 µm 3 QIAxcel PCR µm PCR PCR 1 2 µm PCR PCR Handbook 47 Appendix F Type-it Mutation Detect PCR 06/

18 f g h GC i j k PCR * 20 Handbook 38 Appendix B 3 90 PCR Handbook 47 Appendix F 0.5 x Q-Solution PCR Handbook 36Appendix A GC 1x Q-Solution PCR PCR Handbook 38Appendix B 25 µl 300 ng 25 µl 200 ng DNeasy Kit DNA * MSDS material safety data sheet 18 Type-it Mutation Detect PCR 06/2008

19 l m PCR n o p a Handbook 40Appendix C PCR 0.5 kb 0.5 kb 30 3 PCR QIAxcel Agilent 2100 Bioanalyzer PCR 10 PCR 1.5 kb PCR PCR * 20 * MSDS material safety data sheet Type-it Mutation Detect PCR 06/

20 b c PCR d e 0.2 µm µm 3 QIAxcel PCR PCR µm Handbook 40 Appendix C PCR Handbook 47 Appendix F QIAxcel Agilent 2100 Bioanalyzer PCR 10 PCR 1.5 kb PCR PCR Handbook 47 Appendix F 20 Type-it Mutation Detect PCR 06/2008

21 f GC g 3 PCR PCR a PCR b PCR c d Mg 2+ Q-Solution PCR GC 1x Q-Solution PCR PCR Handbook 47 Appendix F Handbook 38Appendix B 2 90 PCR Handbook 47Appendix F Type-it Multiplex PCR Master Mix 3 mm Mg 2+ Mg 2+ Mg mm Mg 2+ PCR Type-it Mutation Detect PCR 06/

22 e f g PCR h i 0.2 µm µm 3 QIAxcel PCR PCR µm Handbook 40 Appendix C PCR Handbook 38 Appendix B Handook 47 Appendix F PCR * 20 PCR PCR Handbook 38 Appendix B * MSDS material safety data sheet 22 Type-it Mutation Detect PCR 06/2008

23 j GC PCR * PCR a PCR b c d GC PCR e Q-Solution PCR GC 1x Q-Solution PCR PCR 3 DNA Handbook 43 Appendix C Table 16 DNA 25 µl 300 ng PCR PCR 10 PCR 1.5 kb PCR PCR 1.5 kb PCR GC PCR * MSDS material safety data sheet Type-it Mutation Detect PCR 06/

24 . PCR PCR a b stutter peaks c d n 1 e PCR PCR ABI xl DNA Analyzer 10,000 DNA 1 stutter peak 1 n PCR PCR 24 Type-it Mutation Detect PCR 06/2008

25 a PCR b Handbook 38 Appendix B Q- Solution a b PCR PCR 1 2 PCR µm 10 2 µm O- 100 µs/cm Type-it Mutation Detect PCR 06/

26 Type-it Mutation Detect PCR 06/2008

27 Memo Type-it Mutation Detect PCR 06/

28 Trademarks: QIAGEN, QIAamp, CoralLoad, DNeasy, HotStarTaq, REPLI-g, Q-Solution (QIAGEN Group); ABI, ABI PRISM, Applied Biosystems (Applera Corporation or its subsidiaries); Agilent (Agilent Technologies, Inc.); CEQ (Beckman Coulter, Inc.); PAXgene (PreAnalytiX GmbH); LabChip (Caliper Life Sciences, Inc.); Operon (Operon Biotechnologies). QIAGEN 2008 QIAGEN, all rights reserved Forefront Tower II Tel: Fax: /2008 Sample & Assay Technologies

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