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  • Universal USER Cassette Protocol 2: USER-Friendly Vector Preparation

    Overview

    Any standard plasmid DNA purification can be used as long as a phenol/chloroform extraction and subsequent ethanol precipitation step are employed. We have found that Qiagen DNA column purifications may not be completely digested unless the DNA is phenol/chloroform extracted and ethanol precipitated. This protocol is for 10 µg DNA but can, of course, be scaled up. All parts of the protocol should be followed exactly.

    Protocol

    1. Digest plasmid with Xba I restriction endonuclease (NEB #R0145):
      Plasmid DNA (at least 640µg/ml) 10 µg
      NEBuffer 4 10 µl
      BSA (10 mg/ml) 1 µl
      XbaI(40 units) 2 µl
      H2O to 100 µl
      Incubate overnight at 37°C.
    2. Nick the XbaI-linearized vector with Nt.BbvCI (NEB #R0632) by adding 2 µl (20 units) of Nt.BbvCI (10,000 units/ml) to the above reaction and incubating for 1 hour at 37°C.
    3. Purify linearized, nicked vector by phenol-chloroform extraction. Resuspend in 100 µl of TE buffer.
    4. Determine vector concentration. Dilute to 20 µg/ml final concentration as an alternative to the pNEB206A cut vector in the USER Friendly Cloning Kit Protocol.