Answer to Question 2 from 1993

Note that the protruding end left by Bcl I cleavage, 'GATC (note: ' indicates the cleavage position), is identical to that left by Bam HI cleavage. Similarly, the protruding end left by Sal I cleavage, 'TCGA, is identical to that produced by cutting with Xho I. Consequently, the optimum strategy for subcloning the ESP gene is to cut the cosmid DNA with Bcl I and Sal I, use gel electrophoresis to purify the Bcl I-Sal I fragment containing the ESP gene on the basis of its size, cut the vector DNA with Bam HI and Xho I, and then use standard cohesive end ligation techniques to insert the ESP gene fragment into the vector between the Bam HI and Xho I sites. The final ESP-gene-containing circular DNA molecule will no longer have a Bcl I, BamH I, Sal I or Xho I site. Why? It will contain only one Eco RV site. Why?

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