Team:Chiba/jk/γ/week 5

From 2008.igem.org

Week 5

14,September,2008

[http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510]
[http://partsregistry.org/Part:BBa_I716463 BBa_I716463]
[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]
[http://partsregistry.org/Part:BBa_C0070 BBa_C0070]
[http://partsregistry.org/Part:BBa_J06620 BBa_J06620]
[http://partsregistry.org/Part:BBa_J22141 BBa_J22141]
[http://partsregistry.org/Part:BBa_C0076 BBa_C0076]
BBa_I716542
[http://partsregistry.org/Part:BBa_I716462 BBa_I716462]
[http://partsregistry.org/Part:BBa_C0078 BBa_C0078]
[http://partsregistry.org/Part:BBa_J01101 BBa_J01101]
[http://partsregistry.org/Part:BBa_C0071 BBa_C0071]
[http://partsregistry.org/Part:BBa_I712019 BBa_I712019]
[http://partsregistry.org/Part:BBa_S01416 BBa_S01416]
[http://partsregistry.org/Part:BBa_C0079 BBa_C0079]
[http://partsregistry.org/Part:BBa_C0077 BBa_C0077]
[http://partsregistry.org/Part:BBa_J23007 BBa_J23007]
[http://partsregistry.org/Part:BBa_I7100 BBa_I7100](x2)
[http://partsregistry.org/Part:BBa_S01416 BBa_S01416]

Sample No	each
DNA(digested sample)	3
Buffer2(x10)	1
dH2O	6
EcoRⅠ	0.075
TOTAL	10

-->37℃ 1hour

Gel check

Sample No	each
DNA(digestion sample)	10
Loading Dye	2
TOTAL	12

16,September,2008

Digestion test

[http://partsregistry.org/Part:BBa_R0040 BBa_R0040](ptet)+[http://partsregistry.org/Part:BBa_S03158 BBa_S03158](RBS+RhlR)(x2)
[http://partsregistry.org/Part:BBa_R0040 BBa_R0040](ptet)+[http://partsregistry.org/Part:BBa_S03156 BBa_S03156](RBS+lasR)(x5)
[http://partsregistry.org/Part:BBa_R0040 BBa_R0040](ptet)+[http://partsregistry.org/Part:BBa_S03160 Ba_S03160](RBS+cinR)(x2)
[http://partsregistry.org/Part:BBa_S03158 BBa_S03158](RBS+RhlR)
[http://partsregistry.org/Part:BBa_S03156 BBa_S03156](RBS+lasR)
[http://partsregistry.org/Part:BBa_S03160 Ba_S03160](RBS+cinR)

</tr>

Sample No.	each
DNA	3
EcoRⅠ	0.083
PstⅠ	0.083
BSA(x10)	1
Buffer3(x10)	1
dH2O	5
TOTAL	10

-->37℃ 1hour

Gel check

Sample No.	each
DNA	10
Loading Dye	2
TOTAL	12

Transformation

[http://partsregistry.org/Part:BBa_J37019 BBa_J37019](LuxI+RBS+LuxR)->no colony
[http://partsregistry.org/Part:BBa_J37033 BBa_J37033](RBS+LuxR)
[http://partsregistry.org/Part:BBa_J37020 BBa_J37020](LuxI+LuxR+pLux+GFP)
[http://partsregistry.org/Part:BBa_J37032 BBa_J37032](pLux+GFP)

PCR

Venus YFP(ptet+LuxR+YFP)

Sample No.	YFP
DNA	1
Venus Fwd	5
Rev (VR)	5
Thermo pol Buffer	10
dNTP	10
Vent	1
dH2O	68
TOTAL	100

17,September,2008

Transformation

BBa_I13500(RBS+GFP)
BBa_J37019(Luxpr+LuxR)-->no colony

MINI Prep

BBa_J37033(RBS+LuxR)
BBa_J37020(Luxpr+LuxR+LuxR+GFP)
BBa_J37032(pLux+GFP)

Digestion

BBa_I7100(ptetR+RBS)
BBa_S03119(ptet+LuxR)
Ligation product(ptet+LasR)
Ligation product(ptet+RhlR)
Ligation product(ptet+cinR)

Sample No	each
DNA template	25
EcoRⅠ	1
PstⅠ	1
BSA(x10)	4
EcoRⅠ Buffer	4
dH2O	5
TOTAL	40

-->37℃ 1hour
->Next day

18,September,2008

SAP 1μl
->37℃　1hour
->30min　65℃
Zymo ->25μl
Ligation
vector:BBa_I7100
insert:BBa_S03119(ptet+LuxR),Ligation product(ptet+LasR,ptet+RhlR,ptet+cinR)

Sample No	Vector&insert	Vector
DNA(vector)	3.5	3.5
DNA(insert)	7.5	-
Ligase	1	1
Ligase Buffer	4	4
dH2O	4	4
TOTAL	20	20

TRANSFORMATION
BBa_J54103(RBS+GFP)
BBa_P2006

Gel check
Ligation product(Venus YFP)
BBa_J37033
BBa_J37020
BBa_J37032

Sample No	each
DNA	each
Loading Dye	1
dH2O	4
TOTAL	6

19,September,2008

colony PCR
ptet+LuxR(x16)
ptet+LasR(x7)
ptet+RhlR(x5)
ptet+cinR(x5)

20,September,2008

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