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Team:Evry/Protocols/13
From 2013.igem.org
BioBrick Assembly
Goal
Once you have your constructions on your plasmids, you need to biobrick them before sending them to the registry.
Preparation
Restriction
For restriction you need: H2O, BSA, Restriction Buffer 1,2,3 or 4 (buffer 2 works for all
combinations of EcoRI-HF, XbaI, PstI, SpeI), two restriction enzymes per sample.
- Determine which restrictions enzymes you need. Please refer to openwetware.org
instructions.
- Determine the DNA-concentration of the vector and the insert.
- Prepare restriction mix for all restrictions. Add for each reaction:
- For each reaction:
1. 12,5 μl H2O
2. 5,0 μl Buffer 1,2,3 or 4
3. 0,5 μl BSA
4. 1,0 μl restriction enzyme 1
5. 1,0 μl restriction enzyme 2
o Add 20 μl restriction mix to a PCR-tube.
o Add 1 μg of DNA.
o Add H2O to a total volume of 50μl.
- Incubate for 2 hours at 37°C, followed by an additional 20 minutes at 80°C to inactivate the
restriction enzymes.
Protocol adapted from Thermo Scientific PCR Purification notebook
PCR Purification
- Purify the restriction digest, and then determine the concentration of the purified DNA.For more information, see the PCR purification protocole.
Ligation
1 http://openwetware.org/wiki/Enzyme_selection_for_BioBricks_digest - Prepare ligation mix.Prepare two extra reactions, “No insert” and “No ligase” that will be used as negative controls.
| Per vector/insert | No insert | No ligase | |
|---|---|---|---|
| Ligase | 0,5 μL | 0,5 μL | 0 |
| Vector | |||
| Insert | 0 μL | ||
| Distilled water | |||
| 10X Buffer | 2 μL | 2 μL | 2 μL |
Test
