Team:Paris Saclay/Protocols/BioBrick Assembly
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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:
- x μl of H20 milliQ (complement)
- y μl of buffer (Fast Digest Buffer 10x) (1x final concentration)
- z μl of Part A DNA
- 1 μl of Enzyme A
- 1 μl 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
- If the buffer used has no Loading Dye, put it with 1x final concentration.
- Follow the Electrophoresis Protocol with the following parameters:
- Make a gel 0.8% agarose
- Use 2% of BET concentration
- Use a box with a long height and a double case for the DNA.
- Run the gel at 100V for one hour
- Determine the weight of the (Eppendorf) tube prior to use.
- Minimize UV exposure time to avoid damaging the DNA.
- Put the DNA fragment with a minimal agarose gel into the tube.
Note: Minimize UV exposure time to avoid damaging the DNA.
DNA extration from agarose gels
- Excise DNA fragment / solubilize gel slice
- Determine the weight of the gel slice by the difference of the tube's weight.
- For each 100 mg of agarose gel < 2% add 200 μl Buffer NTI.
- Incubate sample for 5-10 min at 50°C.
- Vortex the sample briefly every 2-3 min until the gel slice is completely dissolved.
- Bind DNA
- Place a NucleSpin Gel and PCR Clean-up Column into a Collection Tube and load up to 700 μl sample.
- Centrifuge for 30 seconds at 11,000 x g. Discard flow-through.
- Load remaining sample if necessary and repeat the centrifugation step.
- Wash silica membrane
- Add 700 μl Buffer NT3 to the column. Centrifuge for 30 s at 11,000 x g. Discard flow-through.
- Dry silica membrane
- Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
- Elute DNA
- Place the column into a new 1.5 ml micro centrifuge tube. Add 15-30 μl Buffer NE and incubate at room temperature (18-25 °C) for 1 min.
- Centrifuge for 1 min at 11,000 x g.
Ligation