Kun Zhang LabNote Oct week1

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Thursday 2007-10-04

Thu.Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing

Summary of reactions:

Reaction SNP PCR primer Primer location Sample
1 rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 gDNA
2 rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 cDNA
3 rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 PGP1 gDNA
4 rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 GM20431 cDNA
5 rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 GC1F cDNA
6 rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 GC1EP cDNA
7 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 PGP1 gDNA
8 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 GM20431 cDNA
9 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 GC1F cDNA
10 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 GC1EP cDNA
11 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 Hues6 gDNA
12 rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 Hues6 cDNA
13 rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 PGP1 gDNA
14 rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GM20431 cDNA
15 rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GC1F cDNA
16 rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GC1EP cDNA

Template: 10ng genomic DNA or 1ng cDNA

' one reaction 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 Amplification curves didn't shot up after 35 cycles, add 10 more cycles, only #1 came up

Repeat the experiment with Thermo-Start Taq DNA Pol

' one reaction 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