"
Team:HokkaidoU Japan/Notebook/Promoter RandomPromtoer-B0034-mRFP1-B0015
From 2013.igem.org
Maestro E.coli
Notebook
RandomPromtoer-B0034-mRFP1-B0015
-
Start
-
PCR
B0034-mRFP1-B0015
100up, 200dn, KOD Plus Neo -
Get K1084007 from stock
-
PCR purification
B0034-mRFP1-B0015 -
Digestion
Cut K1084007 with SpeI, PstI (using 10×H buffer)
Cut B0034-mRFP1-B0015 with XbaI, PstI (using 10×M buffer) -
Ligation
Ligate K1084007 with B0034-mRFP1-B0015 -
Transformation
K1084007-B0034-mRFP1-B0015
1µL DNA to DH5α -
Colony PCR
K1084007-B0034-mRFP1-B0015 -
PCR
K1084007(colony)
ranori-f, 200dn, KOD FX Neo
We got random promter.
B0034-LacZα-B0015(colony)
100up, 200dn, KOD FX Neo -
Digestion
Cut random promoter with PstI, SpeI (using 10×H buffer)
Cut B0034-LacZα-B0015 with XbaI, PstI (using 10×M buffer) -
Ligation
Ligate random promoter with B0034-LacZα-B0015 -
Transformation
1µL DNA to JM109
to X-Gal plate -
Culture
random promoter-B0034-LacZα-B0015
to X-Gal 50mg/mL, X-Gal 5mg/mL and LBC
We got some blue colonies with different levels of blue. -
Culture
random promoter-B0034-LacZα-B0015 ×10 samples
random promoter-B0034-mRFP1-B0015 ×9 samples
K1084007-B0034-mRFP1-B0015 -
Mini-prep
random promoter-B0034-LacZα-B0015 ×10 samples
random promoter-B0034-mRFP1-B0015 ×9 samples
K1084007-B0034-mRFP1-B0015 -
Culture
random promoter-B0034-mRFP1-B0015
K1084007-B0034-mRFP1-B0015
random promoter-B0034-mRFP1-B0015 ×20 samples -
PCR
random promoter-B0034-mRFP1-B0015 ×2 samples
random promoter-B0034-LacZα-B0015 ×2 samples
100up, 200dn, KOD FX Neo -
PCR
J23112, J23103, J23117, J23106, J23111, J23100
100up, 200dn, KOD FX Neo -
Digestion
Cut (J23112, J23103, J23117, J23106, J23111, J23100) with EcoRI, PstI (using 10×H buffer)
Cut B0034-mRFP1-B0015 with EcoRI, PstI (using 10×H buffer) -
Antarctic phophatase
B0034-mRFP1-B0015 -
Ligation
Ligate (J23112, J23103, J23117, J23106, J23111, J23100) with B0034-mRFP1-B0015 -
Transformation
(J23112, J23103, J23117, J23106, J23111, J23100)-B0034-mRFP1-B0015
1µL DNA to JM109
pre culture -
Culture
(J23112, J23103, J23117, J23106, J23111, J23100) with B0034-mRFP1-B0015
We picked up some colonies to a X-Gal LBC plate. We confirmed that colonies were white. -
Transformation
R0040-B0034-mRFP1-B0015 -
Culture
(J23112, J23103, J23117, J23106, J23111, J23100, R0040, R0010)-B0034-mRFP1-B0015 -
Transformation
random promoter-B0034-mRFP1-B0015
1µL DNA to JM109 to X-Gal plate -
PCR & PCR purification
J23112, J23103, J23117(colony)
100up, 200dn, KOD FX Neo -
Digestion
Cut J23112, J23103, J23117 with EcoRI, PstI (using 10×H buffer)
Cut random promoter-B0034-LacZα-B0015 on pSB1C3 with EcoRI, PstI (using 10×M buffer) -
Antarctic phosphatase
pSB1C3 -
Ligation
Ligate (J23112, J23103, J23117) with pSB1C3 -
Transformation
1µL DNA to JM109 to 10 X-Gal plates -
Culture
We cultured some colonies from Jul 19 and 23 plates to X-Gal plate. -
Assay
Assay for RFP expression -
Culture
random promoter-B0034-mRFP1-B0015 -
Mini-prep
random promoter-B0034-mRFP1-B0015(cultured Aug 8) -
Complete!
