Knight:DNA ligation using NEB Quick Ligation Kit

From OpenWetWare

Revision as of 13:22, 21 December 2007 by Reshma P. Shetty (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Materials

  • We use the Quick Ligation Kit from NEB
  • Deionized, sterile H2O
  • Purified, linearized vector (in H2O)
  • Purified, linearized insert (in H2O)

Ligation Mix

This is the same protocol that NEB recommends.

  • X μL vector (equivalent to 50 ng)
  • Y μL insert (3-fold molar excess, see below)
  • 10 μL 2X Ligase Buffer
  • (10 - X - Y) μL deionized H2O
  • 1 μL Quick Ligase

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}

Procedure

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 10 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 1 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 1 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Incubate 5 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.
Personal tools