"
August/11 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 25: | Line 25: | ||
<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>4</td><td>dH<sub>2</sub>O</td><td>11</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 39: | Line 39: | ||
<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>11</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 06:23, 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)
1-23L 100>
1-15N 10
2-6P 10<
1-6I 10<
1-12H 10
1-18C 10<
1-20F ×
inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
2.digestion and ligation
digestion with restriction enzyme
K204001
| Vector | Insert | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M) | 12 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M) | 5 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| total | 20uL | total | 20uL |
K204002
| Vector | Insert | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L) | 12 | [http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O) | 5 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| 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.Transformation
to get new plasmid
each 4uL(DNA) , 1-14F : 2uL
1-14H kan
1-14F kan
2-18F Amp ('8/10competent cell
1-23J Amp
1-12C Amp (super competent cell??
