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Team:HokkaidoU Japan/Notebook/RBS SD2,4,6-1C3-Replacement
From 2013.igem.org
Maestro E.coli
Notebook
SD2, 4, 6-1C3 Replacement
-
Start
-
Culture
1C3 vector -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn
faild -
Mini-prep
1C3 vector (7/17 cultured) -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn
faild -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn
faild -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn
SD2 faild -
PCR
R0040-SD2-LacZα-B0015 100up 200dn -
Digestion
Cut R0040-SD(2,4,6)-LacZα-B0015 EcoRI and PstI (using 10×H buffer) -
Ligation
Ligate R0040-SD(2,4,6)-LacZα-B0015 with mini-preped 1C3 vector -
Transformation
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
each of 1µL DNA to DH5α
faild
-
Transformation
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
each of 1µL DNA to DH5α
faild -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn -
Digestion
Cut R0040-SD(2,4,6)-LacZα-B0015 EcoRI and PstI (using 10×H buffer) -
Antarctic phophatase
R0040-SD(2,4,6)-LacZα-B0015 -
Ligation
Ligate R0040-SD(2,4,6)-LacZα-B0015 with pSB1C3 vector -
Transformation
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
each of 1µL DNA to DH5α -
Colony PCR
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3 -
Mini-prep
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3 -
Sequencing
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
100up 200dn
faild -
Sequencing
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
100up 200dn
faild -
PCR
R0040-SD(2,4,6)-LacZα-B0015 100up 200dn -
Digestion
Cut R0040-SD(2,4,6)-LacZα-B001 with EcoRI and PstI (using 10×H buffer) -
Antarctic phophatase
R0040-SD(2,4,6)-LacZα-B0015
-
Ligation
R0040-SD(2,4,6)-LacZα-B0015 with pSB1C3 vector -
Transformation
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
each of 1µL DNA to DH5α -
Sequencing
R0040-SD(2,4,6)-LacZα-B0015 on pSB1C3
100up 200dn -
Complete!
