Kun Zhang LabNote Oct week1: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
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| 1||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 gDNA
| 1(1A)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 gDNA
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|-
| 2||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 cDNA
| 2(1B)||rs3753565||rs3753565_F/rs3753565_R||SnpVer2 A1/A12||Hues6 cDNA
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|-
| 3||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||PGP1 gDNA
| 3(1C)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||PGP1 gDNA
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| 4||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GM20431 cDNA
| 4(1D)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GM20431 cDNA
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| 5||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1F cDNA
| 5(1E)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1F cDNA
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| 6||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1EP cDNA
| 6(1F)||rs2295680||rs2295680_F/rs2295680_R||SnpVer2 A2/B1||GC1EP cDNA
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| 7||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||PGP1 gDNA
| 7(1G)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||PGP1 gDNA
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|-
| 8||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GM20431 cDNA
| 8(1H)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GM20431 cDNA
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| 9||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1F cDNA
| 9(2A)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1F cDNA
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| 10||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1EP cDNA
| 10(2B)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||GC1EP cDNA
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| 11||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 gDNA
| 11(2C)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 gDNA
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| 12||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 cDNA
| 12(2D)||rs6103||rs6103_F/rs6103_R||SnpVer2 A9/B8||Hues6 cDNA
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| 13||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||PGP1 gDNA
| 13(2E)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||PGP1 gDNA
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| 14||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GM20431 cDNA
| 14(2F)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GM20431 cDNA
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| 15||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1F cDNA
| 15(2G)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1F cDNA
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|-
| 16||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1EP cDNA
| 16(2H)||rs1264899||rs1264899_cF/rs1264899_cR||Hues6Ver2c F1/F4||GC1EP cDNA
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95C 15min -> 40 cycles of (94C 30S -> 58C 30S -> 72C 1min) -> 72C 3min<br>
95C 15min -> 40 cycles of (94C 30S -> 58C 30S -> 72C 1min) -> 72C 3min<br>
<b>No reaction shot up at 40 cycles. Added 10 more cycles, only 1A, 1B, 2E came up.</b>
<b>No reaction shot up at 40 cycles. Added 10 more cycles, only 1A, 1B, 2E came up.</b>
<br><b> I think there are several issues in this experiment, including the TE diluted DNA and primers, as well as potential DNA degradation.</b>

Latest revision as of 08:03, 8 October 2007

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.