Prbbbb:fusion assembly v1
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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 |
- 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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References
Example reference
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