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Dephosphorylation of Vector Plasmid.

Dephosphorylation of Vector Plasmid.

Note: 37 C, 56 C, 68 C temperatures are required.
  1. Digest the plasmid with the desired enzyme and collect on preparative gel or from some other source. Be sure it has been NH4Ac/EtOH precipitated at least one time. It is often best to reprecipitate (NH4Ac/EtOH) it even if it is from the DNA stocks and already digested.
  2. Dissolve 10 to 30 µg of the digested plasmid in a 45 µl H2O or bring the DNA sample to this volume with H2O. Add 5 µl 10X CIP buffer (500 mM Tris-HCl pH 9.0, 10 mM MgCl2, 1 mM ZnCl2, 10 mM spermidine.3HCl) and 1 µl containing 0.5 to 2 unit CIP (calf intestinal alkaline phosphotase).
  3. Incubate 15 min at 37 C, 15 min at 56 C, add another 1 µl CIP and incubate 15 min at 37 C, 15 min at 56 C again.
  4. Stop the reaction with 40 µl H2O, 10 µl 10X STE (1 M NaCl, 100 mM Tris- HCl pH 8, 10 mM EDTA), and add 5 µl 10 % SDS: heat to 68 C for 15 min.
  5. Add TE to 315 µl, extract the sample once with CHCl3;phenol, once with CHCl3, and once with ether. NH4Ac/EtOH precipitate it (dump, rinse, wipe, and vacuum dry extra carefully).
  6. Resuspend it in TE (10:2) at the rate of 0.5 µg/µl and freeze at -20 C.

Ligation of Insert DNA with Plasmid DNA

  1. Take 1 to 2 µg of digest insert DNA (i.e. DNA to be cloned) and mix with 1 µg of vector DNA, preferably dephosphorylated, (i.e. plasmid DNA) (or you can digest the two at the time then NH4Ac/EtOH precipitate them), add H2O to 15 µl, heat to 65 C, and cool slowly. Be sure that the "sticky ends" are compatible: usually this means that both the insert and vector were digested with the same enzyme.
  2. Add 4 µl 5X ligation buffer, and 1 µl T4 DNA ligase. Mix and incubate at 56 C (check temperature) for 90 min to overnight.
  3. Dilute with 80 µl TE, add to the DNA list, and freeze at -20 C.