Team:KAIT Japan/Notebook

From 2012.igem.org

(Difference between revisions)
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#72°C(1min)
#72°C(1min)
#4°C(Save)
#4°C(Save)
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#*2~4:35cycle'''→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles) '''
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#*2-4:35cycle'''→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles) '''
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#*gradient:60-61°C(+0.1°C)
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#Add one incubated(100μL)
#Add one incubated(100μL)
#Cultivation(overnight)
#Cultivation(overnight)
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----
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==Date:8/18==
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===Colony PCR===
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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)
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*sterilized water(73.5μL)
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Conditions of the thermal cycler
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#95°C(5min)
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#94°C(30sec)
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#61°C(30sec)
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#71°C(40sec)
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#72°C(1min)
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#4°C(Save)
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#*2-4:30cycle
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#*gradient:57-62°C(+0.1°C)

Revision as of 03:03, 18 August 2012

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Home

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Project

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Parts

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Protocol

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Notebook

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Results

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Safety

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Human Practice

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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)
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