2. The plasmid cloning vector Pbr322 (please refer to the figure in the lecture

Question

2. The plasmid cloning vector Pbr322 (please refer to the figure in the lecture slide) is cleaved with the restriction endonuclease PstI. The cloned DNA fragment cut by PstI is 1,000 bp long and has an EcoRI site 250 bp from one end. Two different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis (Lane 1 and 2), giving the patterns shown below. Note that in pBR322, the PstI and EcoRI restriction sites are about 750bp apart. The entire plasmid with no cloned insert has 4,361 bp. The size markers in lane 4 have the number of nucleotides noted on the right. Determine the correct size of nucleotide in DNA fragments A, B, and C. (15 pt

Explanation / Answer

pBR322 size = 4361 bp
Insert size = 1000 bp

Insert is cloned into Pst I site. So, it can ligate in both the directions.
Insert also has an Eco RI site at 250 bp position.

If the insert is ligated in one orientation. Eco RI digestion would produce two bands of the sizes 1000 bp and 4361 bp. This is represented in lane B.

If the insert is ligated in opposite orientation. Eco RI digestion would produce two bands of the sizes 1250 bp and 4111 bp. This is represented in lane A.

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