June 2008 Type-it Mutation Detect PCR PCR SNPs 2 3 QIAxcel Agilent 2100 Bioanalyzer 8 13 17 Sample & Assay Technologies
MSDS material safety data sheet Operon Biotechnologies www.operon.com TE 100 µm 20 2 Type-it Mutation Detect PCR 06/2008
Type-it Mutation Detect PCR Kit HPLC TE10 mm Tris 1 mm EDTA ph 8.0 50 100 µm4 3 PCR PCR PCR ABI PRISM 3100 Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3730 3730xl DNA Analyzer PCR PCR HPLC Handbook 40 45 Appendix C Appendix D PCR PCR PCR 3 4 TE 20 Type-it Mutation Detect PCR 06/2008 3
3. 10x 2 µm * 50 µm 50 pmol/µl 100 µm 100 pmol/µl 20 µl 10 µl TE 500 µl 500 µl * 12 50 µm 25 100 µm 10x Type-it Mutation Detect PCR QIAxcel Agilent 2100 Bioanalyzer 3 5 bp 1 PCR PCR PCR Type-it Mutation Detect PCR Kit 4 5 6 5 6 4 Type-it Mutation Detect PCR 06/2008
4. PCR PCR >200 bp 2,000 bp 1.3 >100 200 bp 1,000 bp 1.4 1.6 >50 100 bp 750 bp 1.7 2.0 20 50 bp 500 bp 2.5 3.0 <20 bp* 250 bp 3.0 4.0 * 20 bp PCR 20 bp MetaPhor agarose FMC Bioproducts www.cambrex.com 500 bp 5. QIAxcelPCR QIAxcel QX DNA High 15 bp 5 kb 100 500 bp Resolution Cartridge 3 5 bp 500 bp 1 kb 50 bp 1 5 kb 200 500 bp QX DNA Screening PCR 15 bp 5 kb 100 500 bp Cartridge 20 50 bp 500 bp 1 kb 50 100 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. PCR Agilent 2100 Bioanalyzer DNA LabChip Kit 1000 25 1,000 bp 100 500 bp 5 10 500 1,000 bp 10 7500 100 7,500 bp 100 1,500 bp 10 10 * 500 bp PCR Type-it Mutation Detect PCR Kit PCR ABI PRISM 310 3100 Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3730 3730xl 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 www.qiagen.co.jp 6 Type-it Mutation Detect PCR 06/2008
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
QIAxcel Agilent 2100 Bioanalyzer SNaPshot Technology SNPs PCR 90 0.2 µm T m 68 PCR Handbook 38 Appendix B 4 3 10x Q-Solution Handbook 38 Appendix A Q-Solution CoralLoad Dye PCR DNA CoralLoad Dye PCR 95 5 HotStarTaq Plus DNA Polymerase 6 1. 2x Type-it Multiplex PCR Master Mix 20 DNA RNAse CoralLoad Dye Q-Solution 8 Type-it Mutation Detect PCR 06/2008
2. 7 Q-Solution 8 DNA PCR 10 25 µl 7 8 Type-it Multiplex PCR Master Mix 1 1 2x Type-it Multiplex PCR Master Mix 3 mm Mg 2+ 7. 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 3 2.5 µl 1x CoralLoad Dye CoralLoad Dye 10x RNase DNA DNA 300 ng DNA 4 100 ng DNA 25 µl *MgCl 2 3 mm 0.2 µm 0.1 0.3 µm 25 µl 2x Type-it Multiplex PCR Master Mix 1 1 CoralLoad Type-it Mutation Detect PCR 06/2008 9
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 3 2.5 µl 1x CoralLoad Dye CoralLoad Dye 10x Q-Solution, 5x 2.5 µl 0.5x Q-Solution RNase DNA DNA 300 ng DNA 4 100 ng DNA 25 µl *MgCl 2 3 mm 0.2 µm 0.1 0.3 µm 25 µl 2x Type-it Multiplex PCR Master Mix 1 1 CoralLoad 3. PCR PCR 4. PCR DNA 300 ng 12 10 5. 6. PCR 11 9 PCR 95 5 HotStarTaq Plus DNA Polymerase 2 8 20 10 Type-it Mutation Detect PCR 06/2008
9. 2x Type-it Multiplex PCR Master Mix PCR Q-Solution 5 95 HotStarTaq Plus DNA Polymerase 3 30 95 90 60 PCR 60 T m * 60 57 30 72 0.5 kb 0.5 kb 0.5 kb 30 35 35 DNA 12 10 10 68 * T m = 2 x number of [A+T] + 4 x number of [G+C] Type-it Mutation Detect PCR 06/2008 11
10. PCRDNA ng * 100 300 30 35 10 100 35 40 0.1 10 40 45 * Handbook 44 Appendix C Table 17 7. QIAxcel Agilent 2100 Bioanalyzer 5 6 4 5 6 PCR 12 Type-it Mutation Detect PCR 06/2008
PCR 90 0.2 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 6 1. 2x Type-it Multiplex PCR Master Mix 20 DNA Q-Solution 2. 11 DNA PCR 10 25 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 06/2008 13
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 4 100 ng DNA 25 µl *MgCl 2 3 mm 0.2 µm 0.1 0.3 µm 25 µl 2x Type-it Multiplex PCR Master Mix 1 1 3. PCR PCR 4. PCR DNA 200 ng 16 13 5. 6. PCR 15 12 PCR 95 5 HotStarTaq Plus DNA Polymerase 2 8 20 14 Type-it Mutation Detect PCR 06/2008
12. PCR 5 95 HotStarTaq Plus DNA Polymerase 3 30 95 90 60 PCR 60 T m * 60 57 30 72 0.5 kb 0.5 kb 0.5 kb 30 28 28 DNA 16 13 30 60 * 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 06/2008 15
13. PCRDNA ng * 50 200 20 24 10 50 24 28 0.1 10 28 32 * Handbook 44 Appendix C Table 17 7. PCR 6 Handbook 45Appendix D PCR 16 Type-it Mutation Detect PCR 06/2008
PCR a HotStarTaq Plus DNA Polymerase b c d e PCR 6 HotStarTaq Plus DNA Polymerase 95 5 10 14 PCR DNA 0.2 µm 0.1 µm 3 QIAxcel PCR 0.3 0.4 µm PCR PCR 1 2 µm 3 4 3 PCR 10 12 13 16 11 9 15 12 90 PCR Handbook 47 Appendix F Type-it Mutation Detect PCR 06/2008 17
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
l m PCR n o p a Handbook 40Appendix C PCR 0.5 kb 0.5 kb 30 3 PCR 11 9 15 12 60 30 45 QIAxcel Agilent 2100 Bioanalyzer PCR 10 PCR 1.5 kb PCR 68 15 PCR * 20 * MSDS material safety data sheet Type-it Mutation Detect PCR 06/2008 19
b c PCR d e 0.2 µm 10 1 2 µm 3 QIAxcel PCR PCR 0.3 0.4 µm Handbook 40 Appendix C 11 9 15 12 90 PCR Handbook 47 Appendix F 11 9 15 12 60 30 45 QIAxcel Agilent 2100 Bioanalyzer PCR 10 PCR 1.5 kb PCR 68 15 11 9 15 12 90 PCR Handbook 47 Appendix F 20 Type-it Mutation Detect PCR 06/2008
f GC g 3 PCR PCR a PCR b PCR c d Mg 2+ Q-Solution PCR GC 1x Q-Solution PCR 11 9 15 12 90 PCR Handbook 47 Appendix F 3 1 2 Handbook 38Appendix B 2 90 PCR Handbook 47Appendix F Type-it Multiplex PCR Master Mix 3 mm Mg 2+ Mg 2+ Mg 2+ 0.5 mm Mg 2+ PCR Type-it Mutation Detect PCR 06/2008 21
e f g PCR h i 0.2 µm 10 1 2 µm 3 QIAxcel PCR PCR 0.3 0.4 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
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 11 9 15 12 PCR 10 PCR 1.5 kb PCR 68 15 68 15 10 PCR 1.5 kb PCR GC PCR * MSDS material safety data sheet Type-it Mutation Detect PCR 06/2008 23
. PCR PCR a b stutter peaks c d n 1 e PCR PCR ABI 3730 3730xl DNA Analyzer 10,000 DNA 1 stutter peak 1 n 1 95 5 11 9 15 12 45 PCR PCR 24 Type-it Mutation Detect PCR 06/2008
a PCR b Handbook 38 Appendix B Q- Solution a b PCR PCR 1 2 PCR 90 3 10 1 µm 10 2 µm O- 100 µs/cm Type-it Mutation Detect PCR 06/2008 25
95 5 26 Type-it Mutation Detect PCR 06/2008
Memo Type-it Mutation Detect PCR 06/2008 27
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