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  • Universal USER Cassette Protocol 1: Cassette Cloning

    Protocol

    1. Either high concentration T4 DNA Ligase (NEB #M0202T/M ) or the Quick Ligation Kit (NEB #M2200 ) may be used to ligate the Universal USER Cassette into the vector of choice. Follow Step 1a or 1b accordingly:

      1a. High Concentration T4 DNA Ligase Reaction Phosphorylated, blunt-ended Vector 0. 1 pmol (see protocol notes for calculation)
      Universal USER Cassette (2.5 pmol/µl) 2 µl
      10X T4 DNA Ligase Reaction Buffer 2 µl
      Concentrated T4 DNA Ligase 1 µl
      H20 to 20 µl
      Total volume 20 µl
      Incubate 2 hours at room temperature. Proceed to transformation step 2 or store ligation reaction at -20° C.
      -OR-
      1b. Quick Ligation Reaction Phosphorylated, blunt-ended Vector 0. 1 pmol
      (see protocol notes for calculation)
      Universal USER Cassette (2.5 pmol/µl) 2 µl
      2X Quick Ligation Reaction Buffer10 µl
      Quick T4 DNA Ligase 1 µl
      H20 to 20 µl
      Total volume 20 µl
      Incubate 10 min at room temperature. Longer incubation times may reduce ligation efficiency. Proceed to transformation step 2 or store ligation reaction at -20° C.
    2. Transformation of ligation reaction: It is unnecessary to purify the DNA from either the high concentration ligase reaction or the Quick Ligase reaction if E. coli chemically competent cells (CaCl2 or RbCl2 treated cells) are used to recover recombinants. Use 5 µl of ligation reaction for 50 µl of competent cells. If using electrocompetent cells, it is necessary to purify the DNA after either ligation protocol prior to electroporation. We recommend spin column purification.

      Notes:
      Vector Constraints: The starting vector of choice cannot have any pre-existing XbaI sites. XbaI is used to generate compatible ends for the USER PCR.

      Ideally, your vector will have no other Nt.BbvCI sites. If any additional Nt.BbvCI sites are present, but greater than 100 bp from each other, or from the new Nt.BbvCI sites in the Universal USER Cassette, these sites will be nicked but no intervening DNA will be removed. However, long-term storage of such a vector, and repeated freeze/thaws, could result in some decay of thevector due to strand separation, reducing the yield of colonies. Removing the Nt.BbvCI sites from the vector backbone will solve this problem. If the final vector designed has Nt.BbvCI site(s) closer that 100 bp, we strongly recommend removing extraneous Nt.BbvCI site(s) prior to use. It is absolutely necessary to remove these site(s) because the DNA strands are likely to separate.

      Using the recommended 1:50 molar ratio of vector DNA to Universal USER Cassette ensures that a reasonable percentage (20-40%) of the transformants will contain an insertion. Using more or less vector DNA than recommended will result in lower numbers of transformants and fewer transformants with a Universal USER Cassette.

      The digested vector of choice must have blunt phosphorylated ends since the 5? ends of the Universal USER cassette are unphosphorylated. If the researcher prefers to use a dephosphorylated vector, the Universal USER Cassette will need to be phosphorylated prior to ligation. T4 Polynucleotide Kinase (NEB #M0201 ) and T4 DNA Ligase Buffer (NEB #B0202 ) can be used for the phosphorylation reaction of the Universal USER Cassette. See the New England Biolabs catalog or website for the phosphorylation protocol as detailed in the T4 Polynucleotide Kinase product information. After the kinase reaction and inactivation, the amount of Universal USER Cassette used in the ligation reaction should be reduced from 50 fold molar excess to three fold molar excess over vector to reduce the frequency of vectors containing multiple USER cassettes.

      Use 0.1 pmol of cut vector in the ligation reaction. See below for calculation of how many ng of vector is required for 0.1 pmol.

      Converting ng of Vector DNA into pmol: To calculate how much vector (in ng) is necessary for 0.1 pmol, the following equation can be used:
      (# of base pairs of vector) x (650 g/mol base pair) x (109 ng/g) x 10-13 mol (i.e. 0.1 pmol)= x ng.
      Example: How many ng of vector pBR322
      (4361 bp) is required for 0.1 pmol
      (4361 base pairs) x (650 g/mol base pair) x (109 ng/g) x 10-13 mol (i.e. 0.1 pmol)= 283 ng

      Recommended Controls:
      We also encourage performing several controls:
      1) An uncut vector control gives a measure of transformation efficiency.
      2) A cut vector control to which no ligase is added, measures the background of undigested vector.
      3) A cut vector to which no universal USER Cassette was added in the ligation reaction confirms that the ends of the vector were blunt and ligatable.