Knight:DNA ligation using T4 DNA ligase

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Revision as of 13:22, 21 December 2007 by Reshma P. Shetty (Talk | contribs)
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Contents

Materials

  • We use the T4 DNA ligase from NEB
  • Deionized, sterile H2O
  • Purified, linearized vector (in H2O)
  • Purified, linearized insert (in H2O)

Ligation Mix

  • X μL vector (equivalent to ~50 ng, can use less)
  • Y μL insert 1
  • Z μL insert 2
  • 1 μL 10X Ligase Buffer
  • (9.5 - X - Y - Z) μL deionized H2O
  • 0.5 μL T4 DNA ligase
  • Reshma 19:12, 13 December 2007 (CST): I frequently digest 500 ng of each part and the destination vector. Then I use 2 μL vector, 3 μL insert 1 and 3 μL insert 2 where all three linearized fragments have been purified with a Qiagen minelute PCR purification.

Calculating Insert Amount

 \rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng}

(Equimolar ratios may be preferable.)

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 appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 0.5 μL 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 only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Incubate 20 mins on the benchtop.
  7. Place on ice until transformation.
  8. Generally 1 μL of ligation mix is sufficient for either chemical transformation or electroporation. The amount of salt in 1 μL ligation mix should not cause arcing.
  9. Optional Heat-inactivate by incubating at 65°C for 20 mins. Then do a purification step to remove PEG. (See notes on DNA ligation.
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