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Alice:LabNotes/2007-10-15
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===Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing=== Summary of reactions: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Reaction''' | align="center" style="background:#f0f0f0;"|'''SNP''' | align="center" style="background:#f0f0f0;"|'''PCR primer''' | align="center" style="background:#f0f0f0;"|'''Primer location''' | align="center" style="background:#f0f0f0;"|'''Sample''' |- | 1(1A)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 gDNA |- | 2(1B)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 cDNA |- | 3(1C)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||PGP1 gDNA |- | 4(1D)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GM20431 cDNA |- | 5(1E)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1F cDNA |- | 6(1F)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1EP cDNA |- | 7(1G)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||PGP1 gDNA |- | 8(1H)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GM20431 cDNA |- | 9(2A)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1F cDNA |- | 10(2B)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1EP cDNA |- | 11(2C)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 gDNA |- | 12(2D)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 cDNA |- | 13(2E)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||PGP1 gDNA |- | 14(2F)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GM20431 cDNA |- | 15(2G)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1F cDNA |- | 16(2H)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1EP cDNA |- | |} Template: 10ng genomic DNA or 2ng cDNA, use new aliquots of templates/primers/SYBG I diluted in nuclease free H2O. {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''one reaction''' | align="center" style="background:#f0f0f0;"|'''total''' |- | Template ||1||17 |- | 10x Buffer||5||85 |- | 25mM MgCl2||3||51 |- | 10mM dNTP||1||17 |- | 10uM forward primer||1||17 |- | 10uM reverse primer||1||17 |- | 50x SYBG I||0.4||6.8 |- | thermo start Taq||0.25||4.25 |- | H2O||37.35||634.95 |- | |} 95C 3min -> 35 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min <br> <b> The result of the graph was not good since only 2 or 3 reactions shot up. Since the reaction enzyme Thermo Start Taq is a chemically modified DNA polymerase, so it might not work as well as antibody modified DNA polymerase. Therefore, the experiment will be repeated with another enzyme Jumpstart Taq. </b>
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