Alice:LabNotes/2007-11-7

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The sequencing result showed that Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as the peaks of each base pair had overlap, so the experiment on these reactions will be repeated.

Repeat Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing[edit]

Summary of reactions:

Reaction SNP PCR primer Primer location 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
12(2D) rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 Hues6 cDNA
14(2F) rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GM20431 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.

' one reaction total
Template 1 17
10x Buffer 5 85
10mM dNTP 1 17
10uM forward primer + reverse primer 1 17
50x SYBG I 0.4 6.8
Jump Start Tag DNA Polymerase 1 17
H2O 40.6 690.2

95C 3min -> 40 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min -> incubate at 15C forever