Team:Chiba/jk/γ

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17,August 2008
PCR
BBa_E0430①
BBa_E0420②
BBa_J06702③
BBa_S01416④
BBa_I73003⑤
BBa_I73004⑥
BBa_I730026⑦
BBa_I763002⑧

>
Sample No
DNA tamplate 11111111
FW primer 55555555
VR primer 55555555
dNTPmix 1010101010101010
thermo pol buffer 1010101010101010
DNA pol(VENT) 11111111
dH2O 6868686868686868
TOTAL 100100100100100100100100



20,August 2008
TRANSFORMATION
BBa_J52008(Luciferase Renilla)-->colony
BBa_I712019(Luciferase Fire fly)-->no colony
BBa_J332002(LacZ)-->colony
BBa_E2030(Venus YFP)-->not so good
YFP(P-GEX)-->colony
BBa_F2620-->colony
BBa_T9002-->colony



21,August 2008
TRANSFORMATION
BBa_J32007(Lux CDABE)-->no colony
BBa_B0034(RBS)-->colony



22,August 2008
MINI PREP
BBa_J52008(Luciferase)
BBa_F2620
BBa_T9002
BBa_J33202(LacZ)
PUC19
PGEX VENUS YFP(GST fusion)

TRANSFORMATION
PGEX VENUS YFP(GST fusion)


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
Digestion
BBa_R0040(ptet)①
BBa_S03158(RBS+RhlR)②
BBa_S03156(RBS+lasR)③
BBa_S03160(RBS+cinR)④
BBa_R0010(pLac)⑤
BBa_J52008(RBS+rLuc)⑥

Sample No
DNA(digestion sample) 505454451454
SpeⅠ 100010
PstⅠ
TOTAL 12





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