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Kun:LabNotes/ASE/2007-10-4
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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 1ng cDNA {| {{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 |- | 50mM MgCl2||1.5||25.5 |- | 10mM dNTP||1||17 |- | 10uM forward primer||1||17 |- | 10uM reverse primer||1||17 |- | 50x SYBG I||0.4||6.8 |- | iTaq||0.25||4.25 |- | H2O||38.85||660.45 |- | |} 95C 3min -> 35 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min <br> <b>Amplification curves didn't shot up after 35 cycles, add 10 more cycles, only 1A, 2B, 2D came up</b><br> Repeat the experiment with Thermo-Start Taq DNA Pol {| {{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 primer mix||1||17 |- | 50x SYBG I||0.4||6.8 |- | ThermoStart Taq||0.25||4.25 |- | dH2O (from the tap)||38.35||651.95 |- | |} 95C 15min -> 40 cycles of (94C 30S -> 58C 30S -> 72C 1min) -> 72C 3min<br> <b>No reaction shot up at 40 cycles. Added 10 more cycles, only 1A, 1B, 2E came up.</b> <br><b> I think there are several issues in this experiment, including the TE diluted DNA and primers, as well as potential DNA degradation.</b>
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