Team:HokkaidoU Japan/Notebook/Promoter RandomPromtoer-B0034-LacZ-B0015
From 2013.igem.org
Maestro E.coli
Notebook
RandomPromtoer-B0034-LacZ-B0015
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Start
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Culture
K1084007-B0034-lacZα-B0015 on pSB1C3 -
Mini-prep
K1084007-B0034-lacZα-B0015 on pSB1C3 -
PCR & PCR purification
random promoter
random-oripro-f, 200b dn, KOD plus Neo -
Digestion & Gel extraction
Cut random promoter-B0034-lacZα-B0015 with XbaI, PstI (using 10×M buffer)
Cut pSB1C3 with XbaI, PstI (using 10×M buffer) -
EtOH precipitation
random promoter-B0034-lacZα-B0015
pSB1C3 -
Ligation
Ligate random promoter-B0034-lacZα-B0015 with pSB1C3 -
Transformation
random promoter-B0034-lacZα-B0015 on pSB1C3
1µL DNA to DH5α -
Culture
Cultivation time for transformation product was elongated from 18 hrs to 24 hrs.
About 10 blue colonies occurred after the cultivation. -
Transformation
random promoter-B0034-lacZα-B0015 on pSB1C3
5µL DNA to DH5α -
EtOH precipitation
random promoter-B0034-lacZα-B0015 on pSB1C3(ligation product) -
Transformation
random promoter-B0034-lacZα-B0015 on pSB1C3
1µL DNA to DH5α
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Ligation
random promoter-B0034-lacZα-B0015 with pSB1C3 -
Transformation
Each samples were transformed and cultivated at 37°C or 30°C
Ligation 30 min, cultivate at 37°C
Ligation 30 min, cultivate at 30°C
Ligation overnight, cultivate at 37°C
Ligation overnight, cultivate at 30°C -
Correction
random promoter-B0034-lacZα-B0015 on pSB1C3
In 5/20, 5 µL of DNA solution was used for transformation instead of 1 µL of DNA solution for transformation. -
Culture
random promoter-B0034-lacZα-B0015 on pSB1C3 -
Colony PCR
random promoter-B0034-lacZα-B0015
EX-F, PS-R -
Culture
Expression comparison
random promoter-B0034-lacZα-B0015 on pSB1C3 -
Mini-prep
random promoter-B0034-lacZα-B0015 on pSB1C3 -
PCR for sequencing
random promoter-B0034-lacZα-B0015
100b up, 200b dn, KOD Plus Neo -
EtOH for sequencing
random promoter-B0034-lacZα-B0015 -
Sequencing
random promoter-B0034-lacZα-B0015 -
Complete!