Kun Zhang LabNote Oct week2

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Friday 2007-10-12[edit]

Fri.Exp1: Repeat: 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
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.

' 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

Fri.Exp2: Set up PCR using the PGP1VER2 primer set[edit]

cDNA primers on the plate

Forward primer Reverse primer Seq. Primer
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12

gDNA primers on the plate:

Forward primer Reverse primer Seq. Primer
A1 A2 A4 A5 A6 A8 A9 A10 A12
B1 B4 B5 B8 B9 B12
C1 C2 C3 C5 C6 C7 C9 C10 C11
D2 D3 D6 D7 D10 D11
E2 E3 E4 E6 E7 E8 E10 E11 E12
F1 F3 F4 F5 F7 F8 F9 F11 F12
G1 G3 G4 G5 G7 G8 G9 G11 G12
H1 H2 H3 H5 H6 H7 H9 H10 H11

Reaction set up:

' one reaction total
Template 1 each
10x Buffer 5 336
50mM MgCl2 1.5 100.8
10mM dNTP 1 67.2
10uM primer mix 1 each
50x SYBG I 0.4 26.88
iTaq 0.25 16.8
H2O 39.85 2677.92

I ran the 32 amplifications on genomic DNAs using the Eppendorf MasterCycler machine. I also lowered the annealing temperature to 56C.