Team:Paris Saclay/Protocols/BioBrick Assembly

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Revision as of 15:39, 5 August 2014

Contents

BioBrick Assembly

This protocol is based on the original 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

  1. Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
    1. xμl of H20 milliQ (complement)
    2. 5μl of buffer (Fast Digest Buffer 10x)
    3. xμl of Part A DNA
    4. 1μl of Enzyme A
    5. 1μl of Enzyme B
  2. Repeat step 1 with Part B
  3. Mix gently both tubes
  4. Incubate at 37°C for one hour
  5. Store Part B at -20°C.

Segregate process

  1. part A
  2. Follow the Electrophoresis Protocol with the following parameters:
    1. Make a gel 0.8% agarose
    2. Use xxx of BET concentration
    3. Use a box with a long height and a double case for the DNA.
    4. Run the gel at 100V for one hour
  3. minimal exposure of UV light
  4. mass of the ependorf (before / after)

Note: Minimize UV exposure time to avoid damaging the DNA.

DNA extration from agarose gels

  1. Excise DNA fragment / solubilize gel slice
    1. Determine the weight of the (Eppendorf) tube prior to use.
    2. Put the DNA fragment with a minimal agarose gel into the tube.
    3. Determine the weight of the gel slice by the difference of the tube's weight.
    4. For each 100 mg of agarose gel < 2% add 200 μl Buffer NTI.
    5. Incubate sample for 5-10 min at 50°C.
    6. Vortex the sample briefly every 2-3 min until the gel slice is completely dissolved.
  2. Bind DNA
    1. Place a NucleSpin Gel and PCR Clean-up Column into a Collection Tube and load up to 700 μl sample.
    2. Centrifuge for 30 seconds at 11,000 x g. Discard flow-through.
    3. Load remaining sample if necessary and repeat the centrifugation step.
  3. Wash silica membrane
    1. Add 700 μl Buffer NT3 to the column. Centrifuge for 30 s at 11,000 x g. Discard flow-through.
  4. Dry silica membrane
    1. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
  5. Elute DNA
    1. 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.
    2. Centrifuge for 1 min at 11,000 x g.

Ligation