DNA fragment was digested from mouse DNA using HindIII restriction enzyme. The f

Question

DNA fragment was digested from mouse DNA using HindIII restriction enzyme. The fragment is 3 kb and has ATG site on the 5’ end. It also has BamHI restriction site at 1.5 kb position and SacII restriction site at 1 kb position. The fragment was cloned into 5 kb plasmid that has EcoRI site at 12 o’clock position, HindIII site at 1 kb position, SacII at 2.5kb position, BamHI at 4.5 kb and is AmpR. We want to clone the mouse DNA fragment under the promoter located at 0.9 kb on the vector. Assuming that the ligation and transformation worked, how can we differentiate between clones with no insert, insert in a clockwise and counterclockwise positions?

Explanation / Answer

if there is no insert present than digestion with EcoR1 gave us a 5 kb fragment only.

if there is an insert present then we get 8 Kb fragments with EcoR1 digestion.

for checking CW or CCW orientation, we have to digest the plasmid inset with the Sac2 enzyme and EcoR1.

if it is CW then we get fragments of 1 kb, 4.5 kb, and 2.5 kb if it is CCW then we will get 2 kb, 3.5 kb and 2.5 kb fragment.

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