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August/13 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 3: | Line 3: | ||
(plate number)-(location on plate) (no. of colonies) | (plate number)-(location on plate) (no. of colonies) | ||
| - | + | 1-2M 100~ (Amp) | |
| - | + | 2-24G 1? | |
| - | + | 1-12C 10 | |
| - | + | 1-6I 10< | |
| - | + | 2-8I 10< | |
| - | 2-6E | + | 2-6E 10< |
| - | 1-16G | + | 1-16G 10 |
| - | 1-12A | + | 1-12A 10< |
| - | 2-8O | + | 2-8O 10< |
2-11N 10 (Kan) | 2-11N 10 (Kan) | ||
| - | + | 1-18L 50 | |
inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL) | inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL) | ||
| Line 36: | Line 36: | ||
<tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr> | <tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr> | ||
<tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | <tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>6</td><td>dH<sub>2</sub>O</td><td>10</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
| Line 48: | Line 48: | ||
<tr><td>XbaI</td><td>1</td><td>SpeI</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> No.2</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>6</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
| Line 61: | Line 61: | ||
<tr><td>PstI</td><td>1</td><td>PstI</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> No.2 </td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>8</td><td>dH<sub>2</sub>O</td><td>8</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
| Line 74: | Line 74: | ||
<tr><td>PstI</td><td>1</td><td>PstI</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> No.2 </td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>6</td><td>dH<sub>2</sub>O</td><td>8</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
Revision as of 06:25, 23 August 2009
1.before Min prep
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)
1-2M 100~ (Amp)
2-24G 1?
1-12C 10
1-6I 10<
2-8I 10<
2-6E 10<
1-16G 10
1-12A 10<
2-8O 10<
2-11N 10 (Kan)
1-18L 50
inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
after ligation
EpsE + terminator 3 → liquid medium
RBS + LasR(2-8M) 10 → rapid check
2.digestion and ligation
digestion with restriction enzyme
K204003
| Vector | Insert | ||
|---|---|---|---|
| (RBS)1-2M | 10 | cinR(1-14F) | 6 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 10 |
| total | 20uL | total | 20uL |
K204004
| Vector | Insert | ||
|---|---|---|---|
| RBS(1-2M) | 10 | LasR(2-8M) | 10 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 6 |
| total | 20uL | total | 20uL |
K204005
| Vector | Insert | ||
|---|---|---|---|
| 1-18L | 8 | 1-23J | 8 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 8 | dH2O | 8 |
| total | 20uL | total | 20uL |
K204006
| Vector | Insert | ||
|---|---|---|---|
| RBS(1-2M) | 10 | 1-14D | 10 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 6 | dH2O | 8 |
| total | 20uL | total | 20uL |
↓
37°C, 2hr
↓
gel electrophoresis
gel cut
↓
purification by [QIAquick Nucleotide Removal Kit]
↓
ligation
ligation DNA 44 10* buffer 5 ligation 1 total 50uL ↓ 16°C overnight
3.ligation check
3.1. EpsE + terminater
Result of Nanodrop concentration check
(sample No.) (concentration)
2-A 21.5 ng/uL
2-B 15.8 ng/uL
2-C 16.7 ng/uL
↓
digestion with restriction enzyme
| 2-A/2-B/2-C | 8 |
| XbaI | 1 |
| PstI | 1 |
| No.2 | 2 |
| dH2O | 8 |
| total | 20uL |
| 1-23L(vector) | 8 |
| XbaI | 1 |
| No.2 | 2 |
| dH2O | 9 |
| total | 20uL |
↓
37°C over night
3.2. rapid check (RBS + LasR(2-8M))
No.3 and No.8 → to iquid medium(5mL + Amp 5uL)
