"
August/10 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 22: | Line 22: | ||
<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | <tr><td>SpeI</td><td>1</td><td>XbaI</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>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 33: | Line 33: | ||
<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | <tr><td>SpeI</td><td>1</td><td>XbaI</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> | ||
Revision as of 06:23, 23 August 2009
1.Min prep
Result of Nanodrop concentration check
(sample No.) (concentration) 1-6O 35.2 ng/uL 1-18L 41.8 ng/uL 1-10K 39.1 ng/uL
2.make competent cells(with CaCl2)
3.digestion and ligation
digestion with restriction enzyme
| Vector1 | Insert1 | ||
|---|---|---|---|
| 1-23L | 12 | 2-16H | 6 |
| EcoRI | 1 | EcoRI | 1 |
| SpeI | 1 | XbaI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 10 |
| total | 20uL | total | 20uL |
| Vector2 | Insert2 | ||
|---|---|---|---|
| 1-23L | 12 | 3-18O | 5 |
| EcoRI | 1 | EcoRI | 1 |
| SpeI | 1 | XbaI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| total | 20uL | total | 20uL |
↓
failure
digestion with restriction enzyme insert : E+S vector : E+x
4.Transformation
to get new plasmid
1-23L A 1-18C A 1-20F K 1-12H K 1-6I A 2-6P A 1-15N A used '8/10 competent cell
