"

From 2012.igem.org

Home

Project

Parts

Protocol

Notebook

Results

Safety

Human Practice

Team

We are creating now...

Contents 1 Creating parts of Tar methylation region. 1.1 Date:8/9 1.1.1 Colony PCR 1.2 Date:8/11 1.2.1 The purified DNA 1.2.2 PCR Product 1.3 Date:8/13 1.3.1 Confirmed of electrophoresis by PCR product and Ligation of the TA vector 1.4 Date:8/14 1.4.1 Transformation 1.5 Date:8/18 1.5.1 Colony PCR 1.6 Date:8/20 1.6.1 DNA extraction and purification of P.aeruginosa

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:dye 1μL,DNA molecule 2μL,TE buffer 3μL
   • Sample:dye 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:dye 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(+0.1c)

---

Date:8/20

DNA extraction and purification of P.aeruginosa

1. Centrifuge culture medium(6,000rpm,5min,4°C)
2. Remove supernatant,Add saline[0.85%](1.5mL)
3. Centrifuge(6,000rpm,5min,4°C)
4. Add 5mMEDTA 1mL,10%SDS 100μL,proteinase K 50μL