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