Preparation of SmaI-linearized, dephosphorylated double-stranded M13 replicative form cloning vector (10)
(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu)
Several small batches of M13RF double stranded DNA are mixed with buffer, SmaI restriction endonuclease, and calf intestinal alkaline phosphatase (CIAP) and incubated for 2-4 hours at 37degC. The reactions then are pooled, ethanol precipitated, and resuspended in buffer to yield a final concentration of about 10 ng/ul. After characterization to determine the optimal concentration for shotgun cloning ligations and to assess the efficiency of SmaI digestion and CIAP dephosphorylation, aliquots of linearized vector are stored frozen at -70degC.
Protocol
1. Prepare 10-20 tubes with the following:
SmaI from New England Biolabs (141L) and CIAP from Boehringer Mannheim (1097 075). NEB Buffer #4 (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 10 mM dithiothreitol, pH 7.9) included with SmaI from New England Biolabs.
2. Incubate at 37degC for 2-4 hours.
3. Pool the reactions, phenol extract, ethanol precipitate, and resuspend the dried DNA in 10:0.1 TE buffer to a final concentration of 10 ng/ul.
Alternatively: Preparation of M13 vector DNA for ligation M13 vectors should be digested with restriction enzymes as follows:
Incubate at 37¡ C for 2 to 4 hours. Inactivate restriction enzyme by heating at 75°C for 10 minutes. If dephosphorylation of the M13 vector DNA is desired, it may be performed at this point (see dephosphorylation procedure, above). Add 90 µl water to give final concentration of 10 ng./µl; use 5 µl (50 ng) per ligation. The linearized vector may be stored at -20¡ C.