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Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment
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Revision as of 09:33, 25 September 2014
Wrong Plac failed Experiment
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Start
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Get anti-sense vector
pHN1257 Great thanks to N. Nakashima -
Transformation
R0010-B0034-E1010-B0015(pSB6A1) from destribution kit 1µL DNA to JM109 -
Liquid Culture
pSB6A1 -
Mini-prep
pSB6A1 -
PCR & PCR purification
R0040-B0034-E1010-B0015 as RBS XhoI, as mRFP NcoI -
Ehanol precipetation
anti-sense(mRFP) -
Digestion
Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart) Cut anti-sense(mRFP) with NcoI, XhoI (using 10×Cut Smart) -
Gel Extraction & Ethanol precipetation
pHN1257 anti-sense(mRFP) -
Ligation
Ligate anti-sense(mFP) with pHN1257 -
Transformation
anti-sense(mRFP) on pHN1257 5µL DNA to DH5α Turbo -
Colony PCR
anti-sense(mRFP) on pHN1257 -
Liquid Culture
anti-sense(mRFP) on pHN1257 -
Mini-prep
anti-sense(mRFP) on pHN1257 -
Transformation
anti-sense(mRFP) on pHN1257 & pSB6A1 2.5µL each DNA to DH5α Turbo -
Asssay(unsuccess)
Culture anti-sense(mRFP) on pHN1257 & pSB6A1 1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally. -
Assay(unsuccess)
Culture anti-sense(mRFP) on pHN1257 & 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...
