Team:Paris Saclay/Protocols/BioBrick Assembly
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Contents |
BioBrick Assembly
This protocol is based on the original [http://parts.igem.org/Help:Assembly/3A_Assembly 3A Assembly] from iGEM. We modified it to a assembly that places the core of one BioBrick - here called Part A - into another BioBrick - here called Part B.
TODO: Process illustration [Format I and Format II]
Procedure
Enzymes
Note: Manipulate the enzymes with a proper subzero temperature support.
Part A:
- Enzyme A: EcoRI
- Enzyme B: SpeI
Part B with Format I (Part B placed after Part A):
- Enzyme A: EcoRI
- Enzyme B: XbaI
Part B with Format II (Part B placed before Part A):
- Enzyme A: SpeI
- Enzyme B: PstI
Digest reaction
- Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
- xμl of H20 milliQ (complement)
- 5μl of buffer (Fast Digest Buffer 10x)
- xμl of Part A DNA
- 1μl of Enzyme A
- 1μl of Enzyme B
- Repeat step 1 with Part B
- Mix gently both tubes
- Incubate at 37°C for one hour
- Store Part B at -20°C.
Segregate process
- part A
- Follow the Electrophoresis Protocol with the following parameters:
- Make a gel 0.8% agarose
- Use xxx of BET concentration
- Use a box with a such long height and a double case for the DNA.
- Run the gel at 100V for one hour
- minimal exposure of UV light
- mass of the ependorf (before / after)