Alice:LabNotes/2007-11-7: Difference between revisions

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(New page: <b> The sequencing result is out, and the Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as they had too much overlap, so the experiment on these reactions will be repeated. </b> <...)
 
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<b> The sequencing result is out, and the Reaction #1, 2, 3, 12, 14, 16 didn't work as expected, as they had too much overlap, so the experiment on these reactions will be repeated. </b> <br>
<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


===Repeat Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing===
Summary of reactions:
Summary of reactions:
Reaction SNP PCR primer Primer location Sample
{| {{table}}
1(1A) rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 gDNA
| align="center" style="background:#f0f0f0;"|'''Reaction'''
2(1B) rs3753565 rs3753565_F/rs3753565_R SnpVer2 A1/A12 Hues6 cDNA
| align="center" style="background:#f0f0f0;"|'''SNP'''
3(1C) rs2295680 rs2295680_F/rs2295680_R SnpVer2 A2/B1 PGP1 gDNA
| align="center" style="background:#f0f0f0;"|'''PCR primer'''
 
| align="center" style="background:#f0f0f0;"|'''Primer location'''
12(2D) rs6103 rs6103_F/rs6103_R SnpVer2 A9/B8 Hues6 cDNA
| align="center" style="background:#f0f0f0;"|'''Sample'''
 
|-
14(2F) rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GM20431 cDNA
| 1(1A)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 gDNA
 
|-
16(2H) rs1264899 rs1264899_cF/rs1264899_cR Hues6Ver2c F1/F4 GC1EP cDNA
| 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.
Template: 10ng genomic DNA or 2ng cDNA, use new aliquots of templates/primers/SYBG I diluted in nuclease free H2O.
' one reaction total
{| {{table}}
Template 1 17
| 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 -> 35 cycles of (95C 15S -> 58C 30S -> 72C 1min) -> 72C 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