Team:KAIT Japan/Notebook

From 2012.igem.org

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<P>[[File:KAIT_Japan2012_logo.png|left|link=Team:KAIT_Japan]][[File:KAIT_Japan2012_Sub.png|right|link=http://www.kait.jp/index2.php]][[File:Kaitjapan_iGEM_official.logo.png‎|right|link=http://ung.igem.org/Main_Page]]
<P>[[File:KAIT_Japan2012_logo.png|left|link=Team:KAIT_Japan]][[File:KAIT_Japan2012_Sub.png|right|link=http://www.kait.jp/index2.php]][[File:Kaitjapan_iGEM_official.logo.png‎|right|link=http://ung.igem.org/Main_Page]]
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!align="center"|[[File:Kaitjapan.home.png|link=Team:KAIT_Japan]]
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[[Team:KAIT_Japan|Home]]
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!align="center"|[[File:Kaitjapan_project.png|link=Team:KAIT_Japan/Project]]
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[[Team:KAIT_Japan/Project|Project]]
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!align="center"|[[File:Kaitjapan_parts.png|link=Team:KAIT_Japan/Parts]]
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!align="center"|[[File:Kaitjapan_protocol.png|link=Team:KAIT_Japan/Protocol]]
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[[Team:KAIT_Japan/Protocol|Protocol]]
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!align="center"|[[File:Kaitjapan_notebook.png|link=Team:KAIT_Japan/Notebook]]
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[[Team:KAIT_Japan/Notebook|Notebook]]
[[Team:KAIT_Japan/Notebook|Notebook]]
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!align="center"|[[File:Kaitjapan_results.png|link=Team:KAIT_Japan/Results]]
!align="center"|[[File:Kaitjapan_results.png|link=Team:KAIT_Japan/Results]]
[[Team:KAIT_Japan/Results|Results]]
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!align="center"|[[File:Kaitjapan_safety.png|link=Team:KAIT_Japan/Safety]]
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[[Team:KAIT_Japan/Safety|Safety]]
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!align="center"|[[File:Kaitjapan_human_practice.png|link=Team:KAIT_Japan/Human_Practice]]
!align="center"|[[File:Kaitjapan_human_practice.png|link=Team:KAIT_Japan/Human_Practice]]
[[Team:KAIT_Japan/Human_Practice|Human Practice]]
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!align="center"|[[File:Kaitjapan_team.png|link=Team:KAIT_Japan/Team]]
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[[Team:KAIT_Japan/Team|Team]]
[[Team:KAIT_Japan/Team|Team]]

Revision as of 01:54, 21 August 2012

KAIT Japan2012 logo.png
KAIT Japan2012 Sub.png
Kaitjapan iGEM official.logo.png


Kaitjapan.home.png

Home

Kaitjapan project.png

Project

Kaitjapan parts.png

Parts

Kaitjapan protocol.png

Protocol

Kaitjapan notebook.png

Notebook

Kaitjapan results.png

Results

Kaitjapan safety.png

Safety

Kaitjapan human practice.png

Human Practice

Kaitjapan team.png

Team


We are creating now...

Contents

Creating parts of Tar methylation region.

Date:8/9

Colony PCR

Reagent

  • TaKaRa Ex Taq(5units/μL) 0.5μL
  • 10×Ex Taq buffer 10μL
  • dNTP Mixture(2.5Meach) 8μL
  • Primer F(10μM) 4μL
  • Primer R(10μM) 4μL
  • Template(E.coli DH5α)

→Reflection:We Took E.coli too many.You should take less E.coli.
Conditions of the thermal cycler

  1. 95°C(5min)
  2. 94°C(30sec)
  3. 61°C(30sec)
  4. 71°C(40sec)
  5. 72°C(1min)
  6. 4°C(Save)
    • 2-4:35cycle→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles)
    • gradient:60-61°C(+0.1°C)

Date:8/11

The purified DNA

  1. Electrophoresis
    • Marker:pigment(buffer) 1μL,DNA molecule 2μL,TE buffer 3μL
    • Sample:pigment(buffer) 1μL,sample 5μL
    • Gel concentration:1.2%,Migration time:30min

→Reflection:Band was less.

  1. Storage

PCR Product

  1. Electrophoresis
    • The gel check and cut
  2. DNA purification
  3. Confirmation of electrophoresis
  4. PCR
    • 50cycle
  5. Storage

Date:8/13

Confirmed of electrophoresis by PCR product and Ligation of the TA vector

  1. Electrophoresis
    • Gel concentration:1.2%,Migration time:30min
    • Marker:Flash Gel 5μL
    • Sample:pigment 1μL,sample 5μL
  2. Check and Colony PCR
  3. Add to TA vector
    • PCR product 2μL
    • pMD20-Tvector 1μL
    • D2W 2μL
    • Ligation Mighty Mix 5μL
  4. Heat insulation(16°C,30min)
  5. Storage(-20°C)

Date:8/14

Transformation

  1. Put competent cells on ice(10~15min)
  2. Add Ligation reaction solution(10μL) and tapping
  3. On the ice(30min)[Transformation]
  4. Add LB medium(0.7mL)
  5. Incubate(60min,37°C)
  6. Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
  7. Add one incubated(100μL)
  8. Cultivation(overnight)

Date:8/18

Colony PCR

Reagent

  • TaKaRa Ex Taq(5units/μL) 0.5μL
  • 10×Ex Taq buffer 10μL
  • dNTP Mixture(2.5Meach) 8μL
  • Primer F(10μM) 4μL
  • Primer R(10μM) 4μL
  • Template(E.coli DH5α)
  • sterilized water(73.5μL)

Conditions of the thermal cycler

  1. 95°C(5min)
  2. 94°C(30sec)
  3. 61°C(30sec)
  4. 71°C(40sec)
  5. 72°C(1min)
  6. 4°C(Save)
    • 2-4:30cycle
    • gradient:57-62°C(+0.1°C)
Retrieved from "http://2012.igem.org/Team:KAIT_Japan/Notebook"