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

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