Kun:LabNote/2007/CpgSeq/2007-10-4: Difference between revisions

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(New page: ===Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing=== Summary of reactions: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Reaction''' | align="center" style="bac...)
 
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===Exp1: PCR on PGP1 and Hues6 gDNA/cDNA for Sanger sequencing===
Summary of reactions:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reaction'''
| align="center" style="background:#f0f0f0;"|'''SNP'''
| align="center" style="background:#f0f0f0;"|'''PCR primer'''
| align="center" style="background:#f0f0f0;"|'''Primer location'''
| align="center" style="background:#f0f0f0;"|'''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
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''one reaction'''
| align="center" style="background:#f0f0f0;"|'''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 <br>
<b>Amplification curves didn't shot up after 35 cycles, add 10 more cycles, only 1A, 2B, 2D 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'''
|-
| 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<br>
<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 02:16, 16 October 2007