Team:Evry/Protocols/13

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Iron coli project

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: distilled water, Bovine Serum Albumin (BSA, used to stabilize restriction enzymes), 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 and Buffer you need using openwetware.org1.
Determine the DNA-concentration of the vector and the insert.
Prepare restriction mix for all restrictions,for each reaction add:

  • 12,5 μl od distilled water
  • 5,0 μl of Buffer 1,2,3 or 4
  • 0,5 μl of BSA
  • 1,0 μl of restriction restriction enzyme 1
  • 1,0 μl of restriction restriction enzyme 2

Then, for each reaction,add:

  • 20 μl restriction mix previously prepared to a PCR-tube
  • 1 μg of DNA.
  • Distilled water, until your reach a total volume of 50 μl
Incubate for 2 hours at 37°C, then put your tubes 20 minutes at 80°C to inactivate the restriction enzymes.

PCR Purification

Purify the restriction digest, and then determine the concentration of the purified DNA with Nanodrop.
For more information, see the PCR purification protocole.

Ligation

Prepare ligation mix.
Prepare two extra reactions, “No insert” and “No ligase” that will be used as negative controls.


For each reaction,add:
  • 50 ng of vector-DNA.
  • Insert in three times molar excess, then add 3·50·(
  • length of insert/length of vector
    • ) ng of insert-DNA.
  • Distilled water, until your reach a total volume of 20 μl
Then, incubate all your tubes for 30 minutes at 16°C.
Note: For “No insert” omit the insert-DNA, for “No ligase” omit the ligase and replace with H2O.
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

Reference:

  1. http://openwetware.org/wiki/Enzyme_selection_for_BioBricks_digest

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