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Team:HokkaidoU Japan/Notebook/Promoter K1084001~010-B0034-lacZ-B0015
From 2013.igem.org
Maestro E.coli
Notebook
K1084001-010-B0034-lacZ-B0015
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Start
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PCR & PCR purification
K1084007-B0034-mRFP1-B0015
100b up, random-oripro-r, KOD plus Neo -
Digestion
Cut random promoter(PCR product) with EcoRI, SpeI(using 10xH buffer)
Cut K1084007-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, SpeI(using 10xH buffer) -
Antarctic phosphatase
pSB1C3 -
Ligation
Ligate random promoter with pSB1C3 -
Transformation
random promoter on pSB1C3
1µL DNA to DH5α -
Colony PCR
random promoter on pSB1C3 -
Culture
K1084007-B0034-mRFP1-B0015 on pSB1C3 -
Mini-prep
K1084007-B0034-mRFP1-B0015 on pSB1C3 -
PCR
K1084007:100b up, oripro-r, KOD plus Neo
pSB1C3:Suf-I-F, XE-R, KOD plus Neo -
EtOH precipitation
K1084007, pSB1C3 -
Digestion
Cut K1084007 with EcoRI, SpeI(using 10xH buffer)
Cut pSB1C3 with EcoRI, SpeI(using 10xH buffer) -
Antarctic phosphatase
K1084007 -
Ligation
Ligate K1084007 with pSB1C3 -
Transformation
K1084007 on pSB1C3
1µL DNA to JM109 -
Transformation
random promoter on pSB1C3
1µL DNA to JM109 -
Colony PCR
random promoter on pSB1C3 -
Ligation
Ligate random promoter with pSB1C3 -
Transformation
random promoter on pSB1C3 -
Colony PCR
random promoter on pSB1C3 -
Sequencing
random promoter on pSB1C3 -
Digestion
Cut I732020 with XbaI, PstI(using 10xM buffer)
Cut R0040-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, PstI(using 10xH buffer) -
Antarctic phosphatase
pSB1C3 -
Ligation
Ligate random promoter with B0034-lacZα-B0015, pSB1C3 -
Transformation
random promoter-B0034-lacZα-B0015 on pSB1C3
5µL DNA to JM109 -
Colony PCR
random promoter-B0034-lacZα-B0015 on pSB1C3 -
Culture
random promoter-B0034-lacZα-B0015 on pSB1C3
random promoter:K1084001~010 -
Sequencing
K1084001~010-B0034-lacZα-B0015
200b dn -
β-galactosidase assay
K1084001~010-B0034-lacZα-B0015 on pSB1C3 -
Complete!
