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Team:KAIT Japan/Notebook
From 2012.igem.org
(Difference between revisions)
| Line 45: | Line 45: | ||
#Storage | #Storage | ||
| - | ===PCR | + | ===PCR Product=== |
#Electrophoresis | #Electrophoresis | ||
#*The gel check and cut | #*The gel check and cut | ||
| Line 57: | Line 57: | ||
==Date:8/13== | ==Date:8/13== | ||
| - | ===Confirmed of electrophoresis by PCR | + | ===Confirmed of electrophoresis by PCR product and Ligation of the TA vector=== |
#Electrophoresis | #Electrophoresis | ||
| + | #*Gel concentration:1.2%,Migration time:30min | ||
| + | #*Marker:Flash Gel 5μL | ||
| + | #*Sample:pigment 1μL,sample 5μL | ||
#Check and Colony PCR | #Check and Colony PCR | ||
#Add to TA vector | #Add to TA vector | ||
| - | #*PCR | + | #*PCR product 2μL |
#*pMD20-Tvector 1μL | #*pMD20-Tvector 1μL | ||
#*D<sub>2</sub>W 2μL | #*D<sub>2</sub>W 2μL | ||
#*Ligation Mighty Mix 5μL | #*Ligation Mighty Mix 5μL | ||
| - | # | + | #Heat insulation(16℃,30min) |
#Storage(-20℃) | #Storage(-20℃) | ||
Revision as of 03:09, 14 August 2012
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During the creation.
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℃(5min)
- 94℃(30sec)
- 61℃(30sec)
- 71℃(40sec)
- 72℃(1min)
- 4℃(Save)
- 2~4:35cycle→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles)
Date:8/11
The purified DNA
- 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.
- 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:pigment 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℃,30min)
- Storage(-20℃)
