Team:Chiba/jk/γ

From 2008.igem.org

(Difference between revisions)
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*[[Team:Chiba/jk/γ/week 4|week 4  (7,September~13,September)]]
*[[Team:Chiba/jk/γ/week 4|week 4  (7,September~13,September)]]
*[[Team:Chiba/jk/γ/week 5|week 5  (14,September~20,September)]]
*[[Team:Chiba/jk/γ/week 5|week 5  (14,September~20,September)]]
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*[[Team:Chiba/jk/γ/week 6|week 6  (21,September~27,Septembe)]]
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*[[Team:Chiba/jk/γ/week 6|week 6  (21,September~27,September)]]

Revision as of 13:38, 25 September 2008

>実験ログ



24,August 2008
TRANSFORMATION
BBa_J63001(yeast optimized YFP)-->colony
Digestion
BBa_F2620(1)①
BBa_F2620(2)②
Menbren Venus YFP③

Sample No
DNA tamplate 333
Buffer3 111
dH2O 666
EcoRⅠ 0.50.50.5
TOTAL 101010


->37℃ 30min

Sample No ①(afetr digestion)②(after digestion)③(afetr digestion)
DNA tamplate 101010
loading Dye 222
TOTAL 121212
Sample No
DNA tamplate 222
loading Dye 111
dH2O 333
TOTAL 666


->135V 30min


25,August,2008
PCR
BBa_E2030①
BBa_J33202②
BL21③
BBa_J52008④
BBa_I712019⑤

>
Sample No
DNA tamplate 11111
FW primer 5(Venus_YFP_fwd)5(LacZ_fwd)5(LacZ_fwd)5(rLUC_fwd)5(fLuc_fwd)
VR primer 5(VR)5(LacZ_rev)5(LacZ_rev)5(VR)5(VR)
dNTPmix 1010101010
thermo pol buffer 1010101010
DNA pol(VENT) 11111
dH2O 6868686868
TOTAL 100100100100100


->Gel

>
Sample No
DNA tamplate 11111
loading Dye 11111
dH2O 44444
TOTAL 66666


MINI prep
Venus
BBa_F2620
TRANSFORMATRION
Venus
PUC19
pGFPUV
pGEX Venus YFP
Membren Venus YFP
PCR
BBa_J63001①

Sample No
DNA tamplate 1
FW primer(Venus) 5
VR primer 5
dNTPmix 10
thermo pol buffer 10
DNA pol(VENT) 1
dH2O 68
TOTAL 100

26,August,2008
Gel check
BBa_F2620
BBa_F2620
BBa_J63001

>
Sample No
DNA tamplate 11111
Loading Dye 11111
dH2O 44444
TOTAL 66666


Digestion
vector:BBa_F2620

</tr>

Sample No BBa_F2620
DNA tamplate 100
SpeⅠ 2
PstⅠ 2
BSA(x10) 13
Buffer2 13
TOTAL 130


insert:BBa_J52008,BBa_E2030

</tr>

Sample No J52008E2030
DNA tamplate 3030
XbaⅠ 11
PstⅠ 11
DpnⅠ 11
Buffer3 44
BSA(x10) 44
TOTAL 4040


->37℃ 3hour
->SAP
->37℃ 30min
->65℃ 15min
->Zymo Clean
Ligation
vector:BBa_F2620 insert:BBa_J52008①
vector:BBa_F2620 insert:BBa_E2030②
Negative control:BBa_F2620③

Sample No
vector 555
inser 107.50
Ligase Buffer 444
Ligase 111
dH2O 02.510
TOTAL 202020


->RT 2hour
->TRANSFORMATION XL10G
->37℃ over night
->colony PCR
->MINI PREP


28,August,2008
TIME RESPONCE(liquid)


29,August,2008
TIME RESPONCE(solid)


30,August,2008
TRANSFORMATION
mcherry
PCR
BBa_J52008(rLuc)

</tr>

Sample No BBa_J52008
DNA tamplate 1
rLuc_fwd 2.5
VR 2.5
thermo pol Buffer 5
dNTP 5
Vent pol 0.5
dH2O 34
TOTAL 50

31,August,2008
digestion
BBa_R0010①
PCR product(BBa_J52008)②

Sample No
DNA 1010
SpeⅠ 10
PstⅠ 11
XbaⅠ 01
BSA(x100) 0.50.5
Buffer2 50
Buffer3 05
dH20 28.528.5
TOTAL 5050


->37℃ 3hour
->①20μl refine
->②20μl refine


1,September,2008
Ligation
vector:BBa_R0010 insert:BBa_J52008①
negative control:BBa_R0010②

</tr>

Sample No
vector 22
insert 30
Ligase Buffer 22
Ligase 11
dH2O 35
TOTAL 1010


->R/T 2hour


TRANSFORMATION
BBa_R0079(Las promoter)
BBa_R0071(RhlR promoter)
BBa_R0077(cinR promoter+RBS)
BBa_R0078(cnR promoter)
BBa_R0062(LUX promoter)



2,September,2008
TIME RESPONCE (Solid)


COLONY PCR
BBa_R0010+BBa_J52008

</tr>

</tr>

Sample No
culture 1
Fwd primer 1.5
Rev primer 1.5
Thermo pol Buffer 3
dNTP mix 3
Taq DNA pol (NEB) 0.2 (1 unit)
dH2O 19.8
TOTAL 30


MINI Prep
BBa_R0079
BBa_R0071
BBa_R0077
BBa_R0078
BBa_R0062


3,September,2008
Digestion test
BBa_R0079①
BBa_R0071②
BBa_R0077③
BBa_R0078④
BBa_R0062⑤

</tr>

Sample No ①~⑤
DNA tamplate 5
Buffer3 1
dH2O 4
EcoRⅠ 0.1
TOTAL 10


->37℃30min
->Gel cheack

Sample No ①~⑤
DNA(digestion sample) 10
loading Dye 2
TOTAL 12

4,Septempber,2008



5,September,2008
TIME RESPONCE(liquid)
Digestion test
BBa_J52008+BBa_F2620
BBa_E2030+BBa_F2620

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


Luciferase cheack->no light


7,September,2008
Cloning
Digestion
vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl

Sample No
DNA(digestion sample) 505454451454
SpeⅠ 100010
PstⅠ 0.50.50.50.50.50.5
XbaⅠ 00.50.50.500.5
BSA(x10) 0.677627
Buffer2 6---2-
Buffer3 -776-7
dH2O 1.91120.51
TOTAL 607070602070


----->37℃ 3hour
----->Gel Extraction

Sample No
DNA(digestion sample) 6101010410
Loading Dye 222212
dH2O 4---12
TOTAL 12121212612
well 1077657


->DNA purification->①-④,⑥->7μl ⑤->6μl


8,Septmeber,2008
SAP
BBa_R0040(ptet)①
BBa_R0010(pLac)⑤

Sample No
DNA(digestion sample) 76
SAP/td> 22
SAP Buffer 33
TOTAL 3030
TOTAL 3030


->37℃ 1hour
->65℃ 30min
->DNA purification
->21μl 6μl
->Gel cheack

Sample No each
DNA(digestion sample) 1
Loading Dye 1
dH2O 4
TOTAL 6


vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl

</tr>

Sample No ①&②①&③①&④⑤&⑥
DNA(vector) 333352.1
DNA(insert) 333-3.1-
Ligase 111111
Ligase Buffer(x5) 222222
dH2O 11148.94
TOTAL 101010102010

9,September,2008


10,September,2008
TIME Responce(liquid)
TIME Responce(solid)
Colony PCR
->Gel cheack
MINI Prep


13,September,2008



14,September,2008
Digestion test
BBa_Q04510
BBa_I716463
BBa_C0051
BBa_C0070
BBa_J06620
BBa_J22141
BBa_C0076
BBa_I716542
BBa_I716462
BBa_C0078
BBa_J01101
BBa_C0071
BBa_I712019
BBa_S01416
BBa_C0079
BBa_C0077
BBa_J23007
BBa_I7100(x2)
BBa_S01416

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


->37℃ 1hour
Gel cheack

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

16,September,2008
Digestion test
BBa_R0040(ptet)+BBa_S03158(RBS+RhlR)(x2)
BBa_R0040(ptet)+BBa_S03156(RBS+lasR)(x5)
BBa_R0040(ptet)+BBa_S03160(RBS+cinR)(x2)
BBa_S03158(RBS+RhlR)
BBa_S03156(RBS+lasR)
BBa_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 cheack

Sample No each
DNA 10
Loading Dye 2
TOTAL 12


TRANSFORATION
BBa_J37019(LuxI+RBS+LuxR)->no colony
BBa_J37003(RBS+LuxR)
BBa_J37020(LuxI+LuxR+pLux+GFP)
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&insertVector
DNA(vector) 3.53.5
DNA(insert) 7.5-
Ligase 11
Ligase Buffer 44
dH2O 44
TOTAL 2020


TRANSFORMATION
BBa_J54103(RBS+GFP)
BBa_P2006


Gel Cheack
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)



22,September,2008
Time delay


23,September,2008
Digestion test
BBa_E0240

Sample No E0240
DNA 2
EcoRⅠ 0.5
PstⅠ 0.5
BSA(x10) 1
EcoRⅠ Buffer(x10) 1
dH2O 5
TOTAL 10


Digestion
Vector:BBa_E0240
Insert:BBa_R0062
Vector:BBa_I7100
Insert:BBa_R0062
Insert:BBa_J37033



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