Team:Chiba/jk/γ
From 2008.igem.org
Line 702: | Line 702: | ||
---- | ---- | ||
'''7,September,2008''' | '''7,September,2008''' | ||
+ | <br>Cloning | ||
<br>Digestion | <br>Digestion | ||
- | <br>BBa_R0040(ptet)① | + | <br>vector:BBa_R0040(ptet)① 75ng/μl |
- | <br>BBa_S03158(RBS+RhlR)② | + | <br>insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl |
- | <br>BBa_S03156(RBS+lasR)③ | + | <br>insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl |
- | <br>BBa_S03160(RBS+cinR)④ | + | <br>insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl |
- | <br>BBa_R0010(pLac)⑤ | + | <br>vector:BBa_R0010(pLac)⑤ 100ng/μl |
- | <br>BBa_J52008(RBS+rLuc)⑥ | + | <br>insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl |
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
<tr> | <tr> | ||
Line 746: | Line 747: | ||
<td>1.9</td><td>1</td><td>1</td><td>2</td><td>0.5</td><td>1</td> | <td>1.9</td><td>1</td><td>1</td><td>2</td><td>0.5</td><td>1</td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
<td>TOTAL</td> | <td>TOTAL</td> | ||
<td>60</td><td>70</td><td>70</td><td>60</td><td>20</td><td>70</td> | <td>60</td><td>70</td><td>70</td><td>60</td><td>20</td><td>70</td> | ||
Line 752: | Line 754: | ||
<br>----->37℃ 3hour | <br>----->37℃ 3hour | ||
<br>----->Gel Extraction | <br>----->Gel Extraction | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①</td><td>②</td><td>③</td><td>④</td><td>⑤</td><td>⑥</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(digestion sample)</td> | ||
+ | <td>6</td><td>10</td><td>10</td><td>10</td><td>4</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Loading Dye</td> | ||
+ | <td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>4</td><td>-</td><td>-</td><td>-</td><td>1</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>12</td><td>12</td><td>12</td><td>12</td><td>6</td><td>12</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>well</td> | ||
+ | <td>10</td><td>7</td><td>7</td><td>6</td><td>5</td><td>7</td> | ||
+ | </table> | ||
+ | <br>->DNA purification->①-④,⑥->7μl ⑤->6μl | ||
+ | ---- | ||
+ | '''8,Septmeber,2008''' | ||
+ | <br>SAP | ||
+ | <br>BBa_R0040(ptet)① | ||
+ | <br>BBa_R0010(pLac)⑤ | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①</td><td>⑤</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(digestion sample)</td> | ||
+ | <td>7</td><td>6</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAP/td> | ||
+ | <td>2</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAP Buffer</td> | ||
+ | <td>3</td><td>3</td><td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td><td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>30</td><td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br>->37℃ 1hour | ||
+ | <br>->65℃ 30min | ||
+ | <br>->DNA purification | ||
+ | <br>->21μl 6μl | ||
+ | <br>->Gel cheack | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>each</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(digestion sample)</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Loading Dye </td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TOTAL</td> | ||
+ | <td>6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br>vector:BBa_R0040(ptet)① 75ng/μl | ||
+ | <br>insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl | ||
+ | <br>insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl | ||
+ | <br>insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl | ||
+ | <br>vector:BBa_R0010(pLac)⑤ 100ng/μl | ||
+ | <br>insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl | ||
+ | <br>Ligation | ||
+ | <table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000"> | ||
+ | <tr> | ||
+ | <td width="257">Sample No</td> | ||
+ | <td>①&②</td><td>①&③</td><td>①&④</td><td>①</td><td>⑤&⑥</td><td>⑤</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(vector)</td> | ||
+ | <td>3</td></td>3</td><td>3</td><td>3</td><td>5</td><td>2.1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA(insert)</td> | ||
+ | <td>3</td><td>3</td><td>3</td><td>-</td><td>3.1</td><td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase</td> | ||
+ | <td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Lidase Buffer(x5)</td> | ||
+ | <td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dH2O</td> | ||
+ | <td>1</td><td>1</td><td>1</td><td>4</td><td>8.9</td><td>4</td> | ||
+ | <td>TOTAL</td> | ||
+ | <td>10</td><td>10</td><td>10</td><td>10</td><td>20</td><td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | ---- | ||
+ | '''9,September.2008''' | ||
+ | <br>TIME RESPONCE(solid) | ||
- | + | --- | |
+ | '''10, September,2008''' | ||
+ | <br>TIME RESPONCE(liquid) | ||
+ | <br>conoly PCR | ||
Revision as of 16:00, 21 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
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 | 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 | |
BSA(x10) | 0.6 | 7 | 7 | 6 | 2 | 7 | |
Buffer2 | 6 | - | - | - | 2 | - | |
Buffer3 | - | 7 | 7 | 6 | - | 7 | |
dH2O | 1.9 | 1 | 1 | 2 | 0.5 | 1 | |
TOTAL | 60 | 70 | 70 | 60 | 20 | 70 |
----->37℃ 3hour
----->Gel Extraction
Sample No | ① | ② | ③ | ④ | ⑤ | ⑥ |
DNA(digestion sample) | 6 | 10 | 10 | 10 | 4 | 10 |
Loading Dye | 2 | 2 | 2 | 2 | 1 | 2 |
dH2O | 4 | - | - | - | 1 | 2 |
TOTAL | 12 | 12 | 12 | 12 | 6 | 12 |
well | 10 | 7 | 7 | 6 | 5 | 7 |
->DNA purification->①-④,⑥->7μl ⑤->6μl
8,Septmeber,2008
SAP
BBa_R0040(ptet)①
BBa_R0010(pLac)⑤
Sample No | ① | ⑤ | |
DNA(digestion sample) | 7 | 6 | |
SAP/td> | 2 | 2 | |
SAP Buffer | 3 | 3 | |
TOTAL | 30 | 30 | |
TOTAL | 30 | 30 |
->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
Ligation
Sample No | ①&② | ①&③ | ①&④ | ① | ⑤&⑥ | ⑤ |
DNA(vector) | 3 | </td>3</td><td>3</td><td>3</td><td>5</td><td>2.1</td>
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