Preparation of SmaI-linearized, dephosphorylated double-stranded M13 replicative form cloning vector

ID: 90

Group:

PCR

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

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.

Year prepared: NULL

Date uploaded: 2010-02-26 14:29:17

url:

Laboratory Protocols