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Team:HokkaidoU Japan/Notebook/Promoter RandomPromtoer-B0034-mRFP1-B0015
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Revision as of 06:53, 27 September 2013
Maestro E.coli
Notebook
oripro(K1084007)-B0034-lacZ-B0015
-
Start
-
Synthesize oripro
Anneal ut-tg-oripro(K1084007)-f and ut-tg-oripro(K1084007)-r -
Culture
pSB1C3 -
EtOH precipitation
K1084007 -
Culture
I732020 -
Mini-prep
pSB1C3, I732020 -
PCR & Gel extraction
I732020
100b up, 200b dn, KOD plus NEO -
Digestion & Gel extraction
Cut pSB1C3 with EcoRI, SpeI (using 10xH buffer) -
EtOH precipitation
pSB1C3 -
Ligation
Ligate pSB1C3 with K1084007 -
Transformation
K1084007 on pSB1C3
5µL DNA to DH5α -
Colony PCR
K1084007 on pSB1C3 -
Mini-prep
K1084007 on pSB1C3 -
PCR for Sequencing
I1084007
100b up, 200b dn -
EtOH for Sequencing
I1084007 -
Sequencing
K1084007 -
Digestion & Gel extraction
Cut I732020 with SpeI, XbaI (using 10xM buffer)
Cut R0040 with SpeI, PstI (using 10xH buffer)
Cut I732020(PCR product) with XbaI, PstI (using 10xM buffer) -
EtOH precipitation
I732020, R0040, I732020(PCR product) -
Ligation(over night)
Ligate K1084007 with I732020
Ligate R0040 with I732020(PCR product) -
Transformation
K1084007-B0034-LacZα-B0015 on pSB1AK3
5µL DNA to DH5α
R0040-B0034-LacZα-B0015 on pSB1A2
5µL DNA to DH5α -
Colony PCR
Èñ©±€æ -
Mini-prep
K1084007-B0034-LacZα-B0015 on pSB1AK3
R0040-B0034-LacZα-B0015 on pSB1A2 -
Culture
K1084007-B0034-LacZα-B0015 on pSB1AK3
R0040-B0034-LacZα-B0015 on pSB1A2
(Add X-Gal to confirm β-galactosidase activity)
X-Gal 50 mg
dimethylformamid 1 mL
(50 mg/mL)
Diluted the X-gal stock 1 to 1 with DW
Pipetted 50 uL of this dilution onto the plate
Evenly distributed the solution onto the plate using a spreader
Dried for 30 min at 37C
Streaked these colonies as "single colony isolation" on each LBA plate -
PCR for Sequencing
K1084007, K1084007-B0034-lacZα-B0015
100b up, 200b dn -
EtOH for Sequencing
K1084007, K1084007-B0034-lacZα-B0015 -
Sequencing
K1084007, K1084007-B0034-lacZα-B0015 -
Culture for assay
K1084007-B0034-lacZα-B0015 on pSB1AK3
R0040-B0034-lacZα-B0015 on pSB1A2
NC:K1084007 on pSB1C3 -
β-galactosidase assay
K1084007-B0034-lacZα-B0015 on pSB1AK3
R0040-B0034-lacZα-B0015 on pSB1A2 -
Digestion & Gel extraction
Cut I732020 with EcoRI, XbaI (using 10xM buffer)
Cut K184007 with EcoRI, SpeI (using 10xH buffer) -
EtOH precipitation
I732020, K1084007 -
Culture
R0040-B0034-mcherry-B0015 on pSB1A2
K1084007 on pSB1C3 -
Mini-prep
R0040-B0034-mcherry-B0015 on pSB1A2
K1084007 on pSB1C3 -
Digestion & Gel extraction
Cut K1084007- B0034-lacZα-B0015 with XbaI, PstI (using 10xM buffer)
Cut K1084007 with XbaI, PstI (using 10xM buffer)
Cut R0040 with XbaI, PstI (using 10xM buffer) -
EtOH precipitation
K1084007-B0034-lacZα-B0015
K1084007
R0040-B0034-mcherry-B0015 -
Ligation
Ligate K1084007-B0034-lacZα-B0015 with pSB1A2
Ligate K1084007-B0034-lacZα-B0015 with pSB1C3 -
Transformation
K1084007-B0034-lacZα-B0015 on pSB1A2
K1084007-B0034-lacZα-B0015 on pSB1C3
5µL DNA to DH5α -
Culture for assay
K1084007-B0034-lacZα-B0015 on pSB1AK3
R0040-B0034-lacZα-B0015 on pSB1A2
NC:K1084007 on pSB1C3 -
β-galactosidase assay
K1084007-B0034-lacZα-B0015 on pSB1AK3
R0040-B0034-lacZα-B0015 on pSB1A2
NC:K1084007 on pSB1C3 -
Complete!
