Restriction endonucleases have used to introdure the phenylalany trna synthetase

Question

Restriction endonucleases have used to introdure the phenylalany trna synthetase gene into the plasmid pREP4 from a commpany called Qiagen, yielding the plasmid called Pro 148. The gene was introduced into the plasmid using the restriction endonuclease Pvull which cuts at the sequence CAG/CTG, and yield blunt ends: CAG and CTG. when you cut the plasmid with Pvull, however, you achieve two fragments that are almost equal in size, and nearly impossible to separate one from the other on an agarose gel. but based on the sequence you find that one fragment cam be further digested with Bgll which recognizes the sequence: GCCNNNNNGGC, yielding the fragments GCCNNNN and NGGC The plasmid is shown below. draw the expected banding pattern one would observe on a 1.0% agarose get, after digestion. from the digestion with Bgll diagram the end of the DNA fragment produced by Bgll digestion.

Explanation / Answer

a. When plasmid is digested with only on restriction enzyme Pvu II since the two fragments generated are of equal size and indistinguishable on the agarose the number of base pairs present in each of the fragments will be approximately half of the number of base pairs present in the plasmid (7765/2 = 3882.5 bp).

The first will contain ORFs

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