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Team:HokkaidoU Japan/Notebook/RBS SD8
From 2013.igem.org
Maestro E.coli
Notebook
SD8
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Start
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Primer Annealing
Anneal rbs-ensd-f and rbs-ensd-r
Anneal B0034-f and B0034-r -
Mini-prep
plasmid extraction of R0040 on pSB1A2 and LacZα on pSB1AK2
However, we forgot to add RNAse to mP1. We failed in mini-prep. cry... -
PCR
B0015 100up, 200dn, KOD Plus NEO -
Mini-prep
plasmid extraction of LacZα on pSB1AK2 -
Digestion
Cut LacZ%alpha; SpeI and PstI(using 10×H buffer), B0015 fragment by XbaI and PstI(using 10×M buffer) -
Ligation
Ligate RBS with R0040 -
Gel Extraction
LacZα -
Ethanol Precipitation
LacZα -
Transformation
R0040-RBS (SD8/ B0034) on pSB1A2
each of 1µL DNA to DH5α
faild -
Digestion
B0015(B0015)
faild -
Digestion
Cut B0015 XbaI and PstI (using 10×M buffer)@ -
Transformation
R0040-RBS (SD8/ B0034) on pSB1A2
each of 1µL DNA to JM109 -
Colony PCR
R0040-(SD8,B0034) -
Culture
R0040-(SD8,B0034) -
Digestion
Cut B0015(B0015) XbaI and PstI (using 10×M buffer) -
Ligation
Ligate LacZαwith B0015 -
Transformation
LacZα-B0015 on pSB1A2
each of 1µL DNA to DH5α -
Colony PCR
R0040-SD8 in JM109 -
Culture
R0040-RBS(SD8x2) -
Mini-Prep
R0040-RBS(B0034) -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
Faild -
Mini-Prep
R0040-RBS(SD8) -
Colony PCR
LacZα-B0015 -
Culture
LacZα-B0015 -
Mini-Prep
LacZα-B0015 -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
faild -
Digestion
Cut R0040-RBS(SD8) SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using 10×M buffer) -
Gel Extraction
R0040-SD8, LacZα-B0015 -
Ethanol Precipitation
R0040-SD8, LacZα-B0015 -
Liquid culture
R0040-B0034 -
Sequencing
R0040-RBS(B0034)
100bup or 200bdn
faild -
Ligation
Ligate R0040-RBS(SD8) with LacZα-B0015 -
Mini-Prep
R0040-B0034 -
Transformation
R0040-RBS(SD8)-LacZα-B0015
each of 5µL DNA to DH5α -
Colony PCR
LacZα-B0015-R0040-RBS(SD8) -
Liquid culture
R0040-SD8 -
Digestion
Cut R0040-SD8 SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using10×M buffer) -
Gel Extraction
R0040-SD8 and LacZα-B0015 -
Ethanol Precipitation
R0040-SD8 and LacZα-B0015 -
Ligation
Ligate R0040-RBS(SD8) with LacZα-B0015 -
Colony PCR
R0040-SD8-LacZα-B0015 on 1A2 in DH5α (yesterday we transformed again) -
Transformation
R0040-SD8-LacZα-B0015 -
Mini-prep
R0040-SD8-LacZα-B0015 -
Sequencing
R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn -
Sequencing
R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn -
β-galactosidase assay (1)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (2)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (3)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (4)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
β-galactosidase assay (5)
R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.) -
Complete!
