From 2010.igem.org
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| | <td>Enzyme</td> | | <td>Enzyme</td> |
| - | <td>1</td> | + | <td>Buffer 1</td> |
| - | <td>2</td> | + | <td>Buffer 2</td> |
| - | <td>3</td> | + | <td>Buffer 3</td> |
| - | <td>4</td> | + | <td>Buffer 4</td> |
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| | <td>NheI</td> | | <td>NheI</td> |
| | + | <td>100</td> |
| | <td>100</td> | | <td>100</td> |
| | <td>10</td> | | <td>10</td> |
Revision as of 17:49, 9 September 2010
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Materials
You will need:
- Sterilized milliQ water
- BSA
- NEB buffers 1,2,3,and/or 4 (see extra notes)
- Miniprepped sample or plasmid you wish to cut
- NEB digestion enzymes (EcoRI, SpeI, PstI, NheI, and/or XbaI)
Extra Notes
Be sure to use the buffer that maximizes the compatibility of the enzyme activity between the two enzymes
| Enzyme |
Buffer 1 |
Buffer 2 |
Buffer 3 |
Buffer 4 |
| EcoRI |
100 |
100 |
100 |
100 |
| SpeI |
75 |
100 |
25 |
100 |
| PstI |
75 |
75 |
100 |
50 |
| NheI |
100 |
100 |
10 |
100 |
| XbaI |
0 |
100 |
75 |
100 |
Procedure
1. Add the following into a PCR tube
- 22μL of milliQ water
- 1μL BSA
- 5μL buffer x (see extra notes)
- 20μL template to be cut
- 1μL digestion enzyme 1
- 1μL digestion enzyme 2
2. Run in a machine
- 37°C for 3 hours
- 80°C for 20 minutes
- Keep at 4°C if to be stored
Purpose
To cut DNA at the designated places; cut out the insert from the plasmid.
References
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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