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Team:KAIT Japan/Notebook
From 2012.igem.org
(Difference between revisions)
| Line 77: | Line 77: | ||
===The purified DNA=== | ===The purified DNA=== | ||
#Electrophoresis | #Electrophoresis | ||
| - | #*Marker: | + | #*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL |
| - | #*Sample: | + | #*Sample:dye 1μL,sample 5μL |
#*Gel concentration:1.2%,Migration time:30min | #*Gel concentration:1.2%,Migration time:30min | ||
'''→Reflection:Band was less.''' | '''→Reflection:Band was less.''' | ||
| Line 99: | Line 99: | ||
#*Gel concentration:1.2%,Migration time:30min | #*Gel concentration:1.2%,Migration time:30min | ||
#*Marker:Flash Gel 5μL | #*Marker:Flash Gel 5μL | ||
| - | #*Sample: | + | #*Sample:dye 1μL,sample 5μL |
#Check and Colony PCR | #Check and Colony PCR | ||
#Add to TA vector | #Add to TA vector | ||
| Line 113: | Line 113: | ||
==Date:8/14== | ==Date:8/14== | ||
===Transformation=== | ===Transformation=== | ||
| - | #Put competent cells on ice(10 | + | #Put competent cells on ice(10-15min) |
#Add Ligation reaction solution(10μL) and tapping | #Add Ligation reaction solution(10μL) and tapping | ||
#On the ice(30min)[Transformation] | #On the ice(30min)[Transformation] | ||
| Line 142: | Line 142: | ||
#4°C(Save) | #4°C(Save) | ||
#*2-4:30cycle | #*2-4:30cycle | ||
| - | #*gradient:57-62°C(+0. | + | #*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,10%SDS 100μL,proteinase K 50μL | ||
| + | # | ||
Revision as of 02:42, 21 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,10%SDS 100μL,proteinase K 50μL
