Kun Zhang LabNote Oct week1: Difference between revisions
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Template: 10ng genomic DNA or | Template: 10ng genomic DNA or 1ng cDNA | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''''' | | align="center" style="background:#f0f0f0;"|'''''' | ||
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95C 3min -> 35 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min | 95C 3min -> 35 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min | ||
<b>Amplification curves didn't shot up after 35 cycles, add 10 more cycles, only #1 came up</b><br> | |||
Repeat the experiment with Thermo-Start Taq DNA Pol | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''one reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''total''' | |||
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| Template||1||17 | |||
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| 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 | |||
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|} | |||
95C 15min -> 40 cycles of (94C 30S -> 58C 30S -> 72C 1min) -> 72C 3min |
Revision as of 01:23, 5 October 2007
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