Team:Chiba/jk/γ
From 2008.igem.org
Line 567: | Line 567: | ||
---- | ---- | ||
'''2,September,2008''' | '''2,September,2008''' | ||
+ | <br>TIME RESPONCE (Solid) | ||
+ | |||
<br>COLONY PCR | <br>COLONY PCR | ||
- | <br>BBa_R0010+ | + | <br>BBa_R0010+BBa_J52008 |
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
<tr> | <tr> | ||
<td width="257">Sample No</td> | <td width="257">Sample No</td> | ||
- | <td>① | + | <td>①</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td></td> | + | <td>culture</td> |
- | <td> | + | <td>1</td> |
+ | </tr> | ||
+ | <td>Fwd primer</td> | ||
+ | <td>1.5</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>Rev primer</td> |
- | <td> | + | <td>1.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>Thermo pol Buffer</td> |
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dNTP mix</td> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Taq DNA pol (NEB)</td> | ||
+ | <td>0.2 (1 unit)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>19.8</td> | ||
+ | </tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br>MINI Prep | ||
+ | <br>BBa_R0079 | ||
+ | <br>BBa_R0071 | ||
+ | <br>BBa_R0077 | ||
+ | <br>BBa_R0078 | ||
+ | <br>BBa_R0062 | ||
+ | |||
+ | ---- | ||
+ | '''3,September,2008''' | ||
+ | <br>Digestion test | ||
+ | <br>BBa_R0079① | ||
+ | <br>BBa_R0071② | ||
+ | <br>BBa_R0077③ | ||
+ | <br>BBa_R0078④ | ||
+ | <br>BBa_R0062⑤ | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①~⑤</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA tamplate</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer3</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRⅠ</td> | ||
+ | <td>0.1</td> | ||
+ | </tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br>->37℃30min | ||
+ | <br>->Gel cheack | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①~⑤</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(digestion sample)</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>loading Dye</td> | ||
<td>2</td> | <td>2</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>TOTAL</td> |
- | <td> | + | <td>12</td> |
</tr> | </tr> | ||
+ | </table> | ||
+ | |||
+ | ---- | ||
+ | '''4,Septempber,2008''' | ||
+ | |||
+ | |||
+ | |||
+ | ---- | ||
+ | '''5,September,2008''' | ||
+ | <br>TIME RESPONCE(liquid) | ||
+ | <br>Digestion test | ||
+ | <br>BBa_J52008+BBa_F2620 | ||
+ | <br>BBa_E2030+BBa_F2620 | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
<tr> | <tr> | ||
- | <td> | + | <td width="257">Sample No</td> |
- | <td> | + | <td>each</td> |
</tr> | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(digestion sample)</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>loading Dye</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
<td>TOTAL</td> | <td>TOTAL</td> | ||
- | <td> | + | <td>12</td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | <br>Luciferase cheack->no light | ||
+ | ---- | ||
+ | '''6,September,2008''' | ||
+ | <br>Digestion | ||
Revision as of 04:31, 17 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
Sample No | BBa_F2620 |
DNA tamplate | 100 |
SpeⅠ | 2 |
PstⅠ | 2 |
BSA(x10) | 13 |
Buffer2 | 13 | TOTAL | 130 |
insert:BBa_J52008,BBa_E2030
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)
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
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
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
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
6,September,2008
Digestion
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 |
---|