Engineering BioBrick vectors from BioBrick parts/DNA ligation

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< Engineering BioBrick vectors from BioBrick parts
Revision as of 22:27, 17 March 2008 by Reshma P. Shetty (Talk | contribs)
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Materials

  • T4 DNA ligase from NEB
  • Deionized, sterile H2O
  • Purified, linearized destination vector (in H2O)
  • Purified, linearized prefix part (in H2O)
  • Purified, linearized suffix part (in H2O)

Ligation Mix

  • 2-4 μL each purified, linearized DNA
  • 1 μL 10X Ligase Buffer
  • 200 units T4 DNA Ligase

deionized H2O to 10μL

Procedure

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 1 μL ligation buffer to the tube.
    Vortex buffer before pipetting to ensure that it is well-mixed.
    Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
  3. Add prefix part, suffix part and destination vector to the tube.
  4. Add T4 DNA ligase.
    Vortex ligase before pipetting to ensure that it is well-mixed.
    Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
  5. Incubate 20 minutes on the benchtop at room-temperature.
  6. Place on ice until transformation.
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