Team:Chiba/jk/γ

From 2008.igem.org

(Difference between revisions)

Revision as of 02:22, 20 September 2008

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

>

Sample No	①	②	③	④	⑤	⑥	⑦	⑧
DNA tamplate	1	1	1	1	1	1	1	1
FW primer	5	5	5	5	5	5	5	5
VR primer	5	5	5	5	5	5	5	5
dNTPmix	10	10	10	10	10	10	10	10
thermo pol buffer	10	10	10	10	10	10	10	10
DNA pol(VENT)	1	1	1	1	1	1	1	1
dH2O	68	68	68	68	68	68	68	68
TOTAL	100	100	100	100	100	100	100	100

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	①	②	③
DNA tamplate	3	3	3
Buffer3	1	1	1
dH2O	6	6	6
EcoRⅠ	0.5	0.5	0.5
TOTAL	10	10	10

->37℃ 30min

Sample No	①(afetr digestion)	②(after digestion)	③(afetr digestion)
DNA tamplate	10	10	10
loading Dye	2	2	2
TOTAL	12	12	12

Sample No	①	②	③
DNA tamplate	2	2	2
loading Dye	1	1	1
dH2O	3	3	3
TOTAL	6	6	6

->135V 30min

25,August,2008
PCR
BBa_E2030①
BBa_J33202②
BL21③
BBa_J52008④
BBa_I712019⑤

>

Sample No	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
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	10	10	10	10	10
thermo pol buffer	10	10	10	10	10
DNA pol(VENT)	1	1	1	1	1
dH2O	68	68	68	68	68
TOTAL	100	100	100	100	100

->Gel

>

Sample No	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
loading Dye	1	1	1	1	1
dH2O	4	4	4	4	4
TOTAL	6	6	6	6	6

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

Sample No	①
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	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
Loading Dye	1	1	1	1	1
dH2O	4	4	4	4	4
TOTAL	6	6	6	6	6

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	J52008	E2030
DNA tamplate	30	30
XbaⅠ	1	1
PstⅠ	1	1
DpnⅠ	1	1
Buffer3	4	4
BSA(x10)	4	4
TOTAL	40	40

->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	①	②	③
vector	5	5	5
inser	10	7.5	0
Ligase Buffer	4	4	4
Ligase	1	1	1
dH2O	0	2.5	10
TOTAL	20	20	20

->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	①	②
DNA	10	10
SpeⅠ	1	0
PstⅠ	1	1
XbaⅠ	0	1
BSA(x100)	0.5	0.5
Buffer2	5	0
Buffer3	0	5
dH20	28.5	28.5
TOTAL	50	50

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

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

</tr>

Sample No	①	②
vector	2	2
insert	3	0
Ligase Buffer	2	2
Ligase	1	1
dH2O	3	5
TOTAL	10	10

->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>

Sample No	①
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	①	②	③	④	⑤	⑥
DNA(digestion sample)	50	54	54	45	14	54
SpeⅠ	1	0	0	0	1	0
PstⅠ	0.5	0.5	0.5	0.5	0.5	0.5
XbaⅠ	0	0.5	0.5	0.5	0	0.5
	TOTAL	12

ホーム	メンバー紹介	プロジェクト紹介	Parts Submitted to the Registry	モデリング	ノート

@@ Line 724: / Line 724: @@
 <tr>
 <td>PstⅠ</td>
+<td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td>
+</tr>
 <tr>
+<td>XbaⅠ</td>
+<td>0</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0</td><td>0.5</td>
+</tr>
+<tr>
+<td>
 <td>TOTAL</td>
 <td>12</td>