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.