Kun Zhang LabNote Oct week1
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Thursday 2007-10-04[edit]
Thu.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 |
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 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 1A, 2B, 2D 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
No reaction shot up at 40 cycles. Added 10 more cycles, only 1A, 1B, 2E came up.
I think there are several issues in this experiment, including the TE diluted DNA and primers, as well as potential DNA degradation.