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August/21 August 2009
From 2009.igem.org
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to get new plasmid | to get new plasmid | ||
| - | ::⑤ | + | ::⑤(RBS+rhlR+terminator+promoter(reg)) |
| + | ::⑦（RBS＋cinR) | ||
| + | ::⑧（RBS＋luxI) | ||
| + | |||
| + | |||
| + | ===check the result of transformation=== | ||
| + | Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies. <br> | ||
| + | |||
| + | (plate number)-(location on plate) (no. of colonies)<br> | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 2-8G] 4 | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2621 2-21F] 0 | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13521 2-6O] 2 | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_E2050 2-13N] <15 | ||
| + | ⑤ 0 | ||
| + | |||
| + | |||
| + | ===restriction and digestion=== | ||
| + | |||
| + | '''K204009''' | ||
| + | <table border="1" Frame="box"> | ||
| + | <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | ||
| + | <tr><td>terminater([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L])</td><td>10</td><td>①-3(RBS+LasR)</td><td>12</td></tr> | ||
| + | <tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr> | ||
| + | <tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr> | ||
| + | <tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| + | <tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>4</td></tr> | ||
| + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | |||
| + | '''K204010''' | ||
| + | <table border="1" Frame="box"> | ||
| + | <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | ||
| + | <tr><td>RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M])</td><td>10</td><td>EpsE+terminater(②-A)</td><td>12</td></tr> | ||
| + | <tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | ||
| + | <tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr> | ||
| + | <tr><td> No.2 </td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| + | <tr><td>dH<sub>2</sub>O</td><td>6</td><td>dH<sub>2</sub>O</td><td>4</td></tr> | ||
| + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | '''K204011''' | ||
| + | |||
| + | <table border="1" Frame="box"> | ||
| + | <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | ||
| + | <tr><td>p(tetR)([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I])</td><td>10</td><td>RBS+GYP+terminater([http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 1-12M])</td><td>12</td></tr> | ||
| + | <tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | ||
| + | <tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr> | ||
| + | <tr><td> No.2 </td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| + | <tr><td>dH<sub>2</sub>O</td><td>6</td><td>dH<sub>2</sub>O</td><td>4</td></tr> | ||
| + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | ↓<br> | ||
| + | 37°C, 2hr<br> | ||
| + | |||
| + | ↓<br> | ||
| + | gel electrophoresis<br> | ||
| + | gel cut<br> | ||
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | '8/22<br> | ||
| + | purification by [QIAquick Nucleotide Removal Kit]<br> | ||
| + | ↓<br> | ||
| + | ligation | ||
| + | ligation | ||
| + | DNA 44 | ||
| + | 10* buffer 5 | ||
| + | ligation 1 | ||
| + | total 50uL | ||
| + | ↓ | ||
| + | 16°C , 4hr | ||
| + | |||
| + | [https://2009.igem.org/Team:Osaka/NOTES back to NOTES] | ||
Latest revision as of 08:01, 12 October 2009
transformation
to get new plasmid
- ⑤(RBS+rhlR+terminator+promoter(reg))
- ⑦（RBS＋cinR)
- ⑧（RBS＋luxI)
check the result of transformation
Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.
(plate number)-(location on plate) (no. of colonies)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 2-8G] 4 [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2621 2-21F] 0 [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13521 2-6O] 2 [http://partsregistry.org/wiki/index.php?title=Part:BBa_E2050 2-13N] <15 ⑤ 0
restriction and digestion
K204009
| Vector | Insert | ||
|---|---|---|---|
| terminater([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]) | 10 | ①-3(RBS+LasR) | 12 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 4 |
| total | 20uL | total | 20uL |
K204010
| Vector | Insert | ||
|---|---|---|---|
| RBS([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 1-2M]) | 10 | EpsE+terminater(②-A) | 12 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 4 |
| total | 20uL | total | 20uL |
K204011
| Vector | Insert | ||
|---|---|---|---|
| p(tetR)([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]) | 10 | RBS+GYP+terminater([http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 1-12M]) | 12 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 4 |
| total | 20uL | total | 20uL |
↓
37°C, 2hr
↓
gel electrophoresis
gel cut
'8/22
purification by [QIAquick Nucleotide Removal Kit]
↓
ligation
ligation DNA 44 10* buffer 5 ligation 1 total 50uL ↓ 16°C , 4hr
