Prbbbb:fusion assembly v1: Difference between revisions
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'''restriction mix B (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>0.25µl</td> <td>5</td> <td>12.5</td> <td>25</td> </tr> | |||
<tr align=right> <td>10 x NEBuffer 1</td> <td>0.5µl</td> <td>10</td> <td>25</td> <td>50</td> </tr> | |||
<tr><td></td></tr> | |||
<tr align=right> <td>EcoRI 20U/µl</td> <td>0.05µl</td> <td>1</td> <td>2.5</td> <td>5</td> </tr> | |||
<tr align=right> <td>AgeI 5U/µl</td> <td>0.2µl</td> <td>4</td> <td>10</td> <td>20</td> </tr> | |||
</table> | |||
# add 1µl DpnI, incubate for 1h @ 37°C | # add 1µl DpnI, incubate for 1h @ 37°C |
Revision as of 11:38, 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 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 | 0.25µl | 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 |
- add 1µl DpnI, incubate for 1h @ 37°C
- purify with PCR purification kit
- elute in water **not** elution buffer
- dilute to standard concentration: 50ng/µl
Notes
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- List troubleshooting tips here.
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References
Example reference
Contact
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