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Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment
From 2014.igem.org
(Difference between revisions)
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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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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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asmRFP(on pHN1257) | asmRFP(on pHN1257) | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | 5µL to DH5α Turbo | ||
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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 | ||
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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 | ||
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</div> | </div> | ||
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| - | 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. | ||
</div> | </div> | ||
Revision as of 03:46, 18 October 2014
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...
