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Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment
From 2014.igem.org
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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> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃ | R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃ | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | KOD plus Neo | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart) | Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart) | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | Cut asmRFP with NcoI, XhoI (using 10×Cut Smart) | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-tile"> | <div class="step-tile"> | ||
<h2 class="step-title"> | <h2 class="step-title"> | ||
| - | Gel Extraction | + | Gel Extraction |
</h2> | </h2> | ||
</div> | </div> | ||
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</div> | </div> | ||
</li> | </li> | ||
| + | <li class="step"> | ||
| + | <div class="step-tile"> | ||
| + | <h2 class="step-title"> | ||
| + | Ethanol precipetation | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div class="step-detail"> | ||
| + | <div class="step-detail-body"> | ||
| + | <ul class="step-detail-trials"> | ||
| + | <li> | ||
| + | <time> | ||
| + | Sun Jul 27 | ||
| + | </time> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="step-recipe"> | ||
| + | pHN1257 & asmRFP | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </li> | ||
| + | |||
<li class="step"> | <li class="step"> | ||
<div class="step-tile"> | <div class="step-tile"> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
asmRFP(on pHN1257) | asmRFP(on pHN1257) | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | 5µL to DH5α Turbo | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃ | asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃ | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | Kapa-Taq | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-recipe"> | <div class="step-recipe"> | ||
asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) | asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | 2.5µL each to DH5α Turbo | ||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | 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. | + | Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | 1. 2mL LB with 100µL 100mM IPTG | ||
| + | 2. 2mL LB However, IPTG was too much to grow normally. | ||
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<div id="footer-content"> | <div id="footer-content"> | ||
<div id="footer-logo"> | <div id="footer-logo"> | ||
| - | <a href="http://igemhokkaidou.com | + | <a href="http://igemhokkaidou.wordpress.com"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a> |
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<div id="footer-twitter"> | <div id="footer-twitter"> | ||
Latest revision as of 15:25, 9 September 2015
Notebook
Lab Documents
Wrong Plac failed Experiment
-
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 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...
