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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 and activity between the two enzymes. The numbers indicate the percentage of enzyme activity for each enzyme in each buffer.
| 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 thermocycler
- 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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