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Restriction Enzyme Based Cloning

Revision as of 14:22, 7 May 2009 by Davebridges (Talk | contribs) (updated with gel purification)

Revision as of 14:22, 7 May 2009 by Davebridges (Talk | contribs) (updated with gel purification)

  1. If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
  2. Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
  3. Gel purify both vector and insert (Qiagen kit).
  • Run out on gel (see Preparing an Agarose Gel
  • On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light
  • Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.
  • Place at 55-65C until gel is dissolved (5-10 min)
  • Add to pink purification column, wash and elute in 30 uL
  1. Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding.
  2. Add 2 uL ligase buffer (single use aliquots).
  3. Add 1 uL T4 DNA Ligase (Invitrogen).
  4. Incubate 2h-O/N at 16C (water bath in cold room).
  5. Transform 5-10 uL into 50 uL supercompetent cells:
    1. Make 50 uL aliquots
    2. Add DNA and mix gently
    3. Incubate on ice for 30min
    4. Heat shock for 45s at 42C
    5. Place on ice for 2min
    6. Add 450 uL LB
    7. Incubate at 37C for 1h with shaking
    8. Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates
  6. Pick several colonies and digest to verify insert. Sequence if necessary.
Last modified on 7 May 2009, at 14:40