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Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | ||
| - | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion# | + | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 02:47, 18 October 2014
Notebook
Lab Documents
Wrong Plac failed Experiment
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Start
-
Get anti-sense vector
pHN1257 Great thanks to N. Nakashima -
Transformation
R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1µL to JM109 -
Liquid Culture
R0010-B0034-E1010-B0015(on pSB6A1) -
Mini-prep
R0010-B0034-E1010-B0015(on pSB6A1) -
PCR & PCR purification
R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃ KOD plus Neo -
Ehanol precipetation
asmRFP -
Digestion
Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart) Cut asmRFP with NcoI, XhoI (using 10×Cut Smart) -
Gel Extraction
pHN1257 & asmRFP -
Ethanol precipetation
pHN1257 & asmRFP -
Ligation
Ligate asmRFP with pHN1257 -
Transformation
asmRFP(on pHN1257) 5µL DNA to DH5α Turbo -
Colony PCR
asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃ Kapa-Taq -
Liquid Culture
asmRFP(on pHN1257) -
Mini-prep
asmRFP(on pHN1257) -
DoubleTransformation
asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) 2.5µL each to DH5α Turbo -
Asssay(unsuccess)
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally. -
Assay(unsuccess)
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20µL 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination. -
Failure...
