Answer to Question 2 of 1994

None of the restriction sites flanking the PBT gene is compatible with the Bam HI or Sal I site in the vector. Fortunately the vector also contains a Sma I site which, after cleavage by Sma I, generates two blunt ends. The optimum cloning strategy is, therefore, very simple. Simply cut the PBT gene out of the cosmid with any pair of restriction enzymes, one from the left of the gene and the other from the right of the gene. The excised gene should be separated from unwanted digestion products by gel electrophoresis. Convert the protruding ends to blunt ends with Klenow polymerase plus dNTPs or with mung bean nuclease, then ligate into the Sma I-cut vector. Transform E. coli  cells, harvest DNA from some of the transformed colonies, and test by restriction digestion and gel electrophoresis that a fragment of the correct size has been cloned into the vector.


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