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Team:KAIT Japan/Notebook
From 2012.igem.org
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===Colony PCR=== | ===Colony PCR=== | ||
Reagent | Reagent | ||
| - | *TaKaRa Ex Taq(5units/μL) 0.5μL | + | :*TaKaRa Ex Taq(5units/μL) 0.5μL |
| - | *10×Ex Taq buffer 10μL | + | :*10×Ex Taq buffer 10μL |
| - | *dNTP Mixture(2.5Meach) 8μL | + | :*dNTP Mixture(2.5Meach) 8μL |
| - | *Primer F(10μM) 4μL | + | :*Primer F(10μM) 4μL |
| - | *Primer R(10μM) 4μL | + | :*Primer R(10μM) 4μL |
| - | *Template(E.coli DH5α) | + | :*Template(E.coli DH5α) |
| - | *sterilized water(73.5μL) | + | :*sterilized water(73.5μL) |
Conditions of the thermal cycler | Conditions of the thermal cycler | ||
#95°C(5min) | #95°C(5min) | ||
Revision as of 01:29, 22 August 2012
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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
- 95°C(5min)
- 94°C(30sec)
- 61°C(30sec)
- 71°C(40sec)
- 72°C(1min)
- 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
- 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.
- Storage
PCR Product
- Electrophoresis
- The gel check and cut
- DNA purification
- Confirmation of electrophoresis
- PCR
- 50cycle
- Storage
Date:8/13
Confirmed of electrophoresis by PCR product and Ligation of the TA vector
- Electrophoresis
- Gel concentration:1.2%,Migration time:30min
- Marker:Flash Gel 5μL
- Sample:dye 1μL,sample 5μL
- Check and Colony PCR
- Add to TA vector
- PCR product 2μL
- pMD20-Tvector 1μL
- D2W 2μL
- Ligation Mighty Mix 5μL
- Heat insulation(16°C,30min)
- Storage(-20°C)
Date:8/14
Transformation
- Put competent cells on ice(10-15min)
- Add Ligation reaction solution(10μL) and tapping
- On the ice(30min)[Transformation]
- Add LB medium(0.7mL)
- Incubate(60min,37°C)
- Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
- Add one incubated(100μL)
- 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
- 95°C(5min)
- 94°C(30sec)
- 61°C(30sec)
- 71°C(40sec)
- 72°C(1min)
- 4°C(Save)
- 2-4:30cycle
- gradient:57-62°C(+0.1c)
Date:8/20
DNA extraction and purification of P.aeruginosa
- Centrifuge culture medium(6,000rpm,5min,4°C)
- Remove supernatant,Add saline[0.85%](1.5mL)
- Centrifuge(6,000rpm,5min,4°C)
- Add 5mMEDTA 1mL
- Add 10%SDS 100μL
Add proteinase K 50μL
- Vortex
- Incubation(30min,55°C)
- Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
- Shake vigorously(1min)
- At this time,It became muddy white in color.
- Centrifuge(16,000rpm,10min,4°C)
- Pick up supernatant,remove new microtube
- Repeat step 7-11
- Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
- Vortex
- Wind the DNA by a thin glass rod.
- Rinse chilled 70%-ethanol(500μL)
- Pick up DNA,air dry
- Add TE buffer 500μL
- Add RNase A 50μL
- Incubation(20min,37°C)
- Add proteinase K 50μL
- Incubation(1h,37°C)
- Add phenol mixture
- Vortex(1min)
- Centrifuge(16,000rpm,10min,4°C)
- Pick up supernatant,remove new microtube
- Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
- Wind the DNA by a thin glass rod.
- Rinse chilled 70%-ethanol(500μL,about 30s)
- Pick up DNA,air dry
- Add TE buffer 200μL
- Melt DNA in buffer
