Prbbbb:fusion assembly v1: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Restriction=== | |||
'''restriction mix A (5x concentrated)''' | '''restriction mix A (5x concentrated)''' | ||
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<td></td> <th width=100>1 µl, single reaction</th> <th width=100>20 µl</th> <th width=100>50 µl</th> <th width=100>100 µl</th></tr> | <td></td> <th width=100>1 µl, single reaction</th> <th width=100>20 µl</th> <th width=100>50 µl</th> <th width=100>100 µl</th></tr> | ||
<tr align=right> <td>H2O</td> <td>0. | <tr align=right> <td>H2O</td> <td>0.2µl</td> <td>4</td> <td>10</td> <td>20</td> </tr> | ||
<tr align=right> <td>10 x NEBuffer | <tr align=right> <td>10 x NEBuffer 4</td> <td>0.5µl</td> <td>10</td> <td>25</td> <td>50</td> </tr> | ||
<tr align=right> <td>100 x BSA</td> <td>0.05</td> <td>0.1</td> <td>2.5</td> <td>5</td> </tr> | |||
<tr><td></td></tr> | |||
<tr align=right> <td>PstI 10U/µl</td> <td>0.125µl</td> <td>2.5</td> <td>6.25</td> <td>12.5</td> </tr> | |||
<tr align=right> <td>NgoMI 10U/µl</td> <td>0.125µl</td> <td>2.5</td> <td>6.25</td> <td>12.5</td> </tr> | |||
</table> | |||
'''restriction mix C (5x concentrated)''' | |||
<table frame=box> | |||
<tr align=right> | |||
<td></td> <th width=100>1 µl, single reaction</th> <th width=100>20 µl</th> <th width=100>50 µl</th> <th width=100>100 µl</th></tr> | |||
<tr align=right> <td>H2O</td> <td>0.2µl</td> <td>4</td> <td>10</td> <td>20</td> </tr> | |||
<tr align=right> <td>10 x NEBuffer EcoRI</td> <td>0.5µl</td> <td>10</td> <td>25</td> <td>50</td> </tr> | |||
<tr align=right> <td>100 x BSA</td> <td>0.05</td> <td>0.1</td> <td>2.5</td> <td>5</td> </tr> | |||
<tr><td></td></tr> | <tr><td></td></tr> | ||
<tr align=right> <td>EcoRI 20U/µl</td> | <tr align=right> <td>EcoRI 20U/µl</td> <td>0.08µl</td> <td>0.16</td> <td>4</td> <td>8</td> </tr> | ||
<tr align=right> <td> | <tr align=right> <td>PstI 10U/µl</td> <td>0.17µl</td> <td>3.4</td> <td>8.5</td> <td>17</td> </tr> | ||
</table> | |||
# mix 8 µl part A [50 ng/µl] with 2 µl restriction A | |||
# mix 8 µl part B [50 ng/µl] with 2 µl restriction B | |||
# mix 8 µl vector [25 ng/µl] with 2 µl restriction C (or use pre-digested stock) | |||
# incubate for 2h @ 37°C | |||
# heat inactivate 20' @ 80°C | |||
===Ligation=== | |||
'''ligation mix (2x concentrated)''' | |||
<table frame=box> | |||
<tr align=right> | |||
<td></td> <th width=100>10 µl, single reaction</th> <th width=100>150 µl</th> <th width=100>µl</th> <th width=100>µl</th></tr> | |||
<tr align=right> <td>H2O</td> <td>7µl</td> <td></td> <td></td> <td></td> </tr> | |||
<tr align=right> <td>5 x T4 buffer</td> <td>2µl</td> <td></td> <td></td> <td></td> </tr> | |||
<tr align=right> <td>T4 DNA Ligase</td> <td>1</td> <td></td> <td></td> <td></td> </tr> | |||
</table> | </table> | ||
# add | # mix 4 µl part A digest + 4 µl part B digest + 2 µl vector digest | ||
# | # add (as last component!) 10 µl ligation mix (2x) | ||
# incubate 1h @ 16°C; 10' @ 65deg;C | |||
# use 2 µl for transformation | |||
==Notes== | ==Notes== | ||
Revision as of 12:15, 11 February 2009
Overview
Materials
- enzymes: AgeI, EcoRI, PstI, NgoMIV (=NgoMI), T4_DNA_Ligase
- NEB_buffers: NEBuffer EcoRI, NEBuffer 1, NEBuffer 4, T4 DNA Ligase buffer
- NEB BSA 100x concentrated
- ddH2O
- linear vector backbone DNA from Prbbbb:vector_pcr; concentration: 25 ng/µl
- DNA Biobrick(s) A (first part), Biobrick(s) B (second part); concentration 50 ng/µl
Procedure
Restriction
restriction mix A (5x concentrated)
| 1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
|---|---|---|---|---|
| H2O | 0.25µl | 5 | 12.5 | 25 |
| 10 x NEBuffer 1 | 0.5µl | 10 | 25 | 50 |
| EcoRI 20U/µl | 0.05µl | 1 | 2.5 | 5 |
| AgeI 5U/µl | 0.2µl | 4 | 10 | 20 |
restriction mix B (5x concentrated)
| 1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
|---|---|---|---|---|
| H2O | 0.2µl | 4 | 10 | 20 |
| 10 x NEBuffer 4 | 0.5µl | 10 | 25 | 50 |
| 100 x BSA | 0.05 | 0.1 | 2.5 | 5 |
| PstI 10U/µl | 0.125µl | 2.5 | 6.25 | 12.5 |
| NgoMI 10U/µl | 0.125µl | 2.5 | 6.25 | 12.5 |
restriction mix C (5x concentrated)
| 1 µl, single reaction | 20 µl | 50 µl | 100 µl | |
|---|---|---|---|---|
| H2O | 0.2µl | 4 | 10 | 20 |
| 10 x NEBuffer EcoRI | 0.5µl | 10 | 25 | 50 |
| 100 x BSA | 0.05 | 0.1 | 2.5 | 5 |
| EcoRI 20U/µl | 0.08µl | 0.16 | 4 | 8 |
| PstI 10U/µl | 0.17µl | 3.4 | 8.5 | 17 |
- mix 8 µl part A [50 ng/µl] with 2 µl restriction A
- mix 8 µl part B [50 ng/µl] with 2 µl restriction B
- mix 8 µl vector [25 ng/µl] with 2 µl restriction C (or use pre-digested stock)
- incubate for 2h @ 37°C
- heat inactivate 20' @ 80°C
Ligation
ligation mix (2x concentrated)
| 10 µl, single reaction | 150 µl | µl | µl | |
|---|---|---|---|---|
| H2O | 7µl | |||
| 5 x T4 buffer | 2µl | |||
| T4 DNA Ligase | 1 |
- mix 4 µl part A digest + 4 µl part B digest + 2 µl vector digest
- add (as last component!) 10 µl ligation mix (2x)
- incubate 1h @ 16°C; 10' @ 65deg;C
- use 2 µl for transformation
Notes
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References
Example reference
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