Alice:LabNotes/2007-11-7: Difference between revisions
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<b> The sequencing result showed that Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as | <b> The sequencing result showed that Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as the peaks of each base pair had overlap, so the experiment on these reactions will be repeated. </b> <br> | ||
===Repeat Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing=== | |||
Summary of reactions: | Summary of reactions: | ||
{| {{table}} | |||
Reaction | | align="center" style="background:#f0f0f0;"|'''Reaction''' | ||
| align="center" style="background:#f0f0f0;"|'''SNP''' | |||
1(1A) rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 gDNA | | align="center" style="background:#f0f0f0;"|'''PCR primer''' | ||
| align="center" style="background:#f0f0f0;"|'''Primer location''' | |||
2(1B) rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 cDNA | | align="center" style="background:#f0f0f0;"|'''Sample''' | ||
|- | |||
3(1C) rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 PGP1 gDNA | | 1(1A)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 gDNA | ||
|- | |||
12(2D) rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 Hues6 cDNA | | 2(1B)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 cDNA | ||
|- | |||
14(2F) rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GM20431 cDNA | | 3(1C)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||PGP1 gDNA | ||
|- | |||
16(2H) rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GC1EP cDNA | | 12(2D)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 cDNA | ||
|- | |||
| 14(2F)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GM20431 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. | Template: 10ng genomic DNA or 2ng cDNA, use new aliquots of templates/primers/SYBG I diluted in nuclease free H2O. | ||
' | {| {{table}} | ||
Template | | align="center" style="background:#f0f0f0;"|'''''' | ||
10x Buffer 5 85 | | align="center" style="background:#f0f0f0;"|'''one reaction''' | ||
10mM dNTP 1 17 | | align="center" style="background:#f0f0f0;"|'''total''' | ||
10uM forward primer + reverse primer 1 17 | |- | ||
50x SYBG I 0.4 6.8 | | Template ||1||17 | ||
Jump Start Tag DNA Polymerase 1 17 | |- | ||
H2O 40.6 690.2 | | 10x Buffer||5||85 | ||
|- | |||
| 10mM dNTP||1||17 | |||
|- | |||
| 10uM forward primer + reverse primer||1||17 | |||
|- | |||
| 50x SYBG I||0.4||6.8 | |||
|- | |||
| Jump Start Tag DNA Polymerase||1||17 | |||
|- | |||
| H2O||40.6||690.2 | |||
|- | |||
| | |||
|} | |||
95C 3min -> | 95C 3min -> 40 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min -> incubate at 15C forever<br> |
Latest revision as of 22:40, 30 November 2007
The sequencing result showed that Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as the peaks of each base pair had overlap, so the experiment on these reactions will be repeated.
Repeat 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 |
12(2D) | rs6103 | rs6103_F/rs6103_R | SnpVer2 A9/B8 | Hues6 cDNA |
14(2F) | rs1264899 | rs1264899_cF/rs1264899_cR | Hues6Ver2c F1/F4 | GM20431 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 |
10mM dNTP | 1 | 17 |
10uM forward primer + reverse primer | 1 | 17 |
50x SYBG I | 0.4 | 6.8 |
Jump Start Tag DNA Polymerase | 1 | 17 |
H2O | 40.6 | 690.2 |
95C 3min -> 40 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 3min -> incubate at 15C forever