11. You are pleased because you think you\'ve obtained a positive clone of pGEM-

Question

11. You are pleased because you think you've obtained a positive clone of pGEM-T:AraC/GFP. You perform diagnostic restriction enzyme digests on purified plasmid from your clone to determine the insert orientation and obtain the following results. Plasmid files are at the 1. Laddert 2. Noenzyme 3. Scal 4. Xhot 5. Scal/xhol end of the exam. (2 points) Circle the band(s), if any, that represents supercoiled plasmid DNA a. 8000 6000 5500 b. (2 points) Box the band(s),if any, that represents linearized400 plasmid DNA 2000 (2 points) Your labmate points out that one of the digest results is unexpected. Do you agree? If so, identify the relevant digest and propose a brief hypothesis accounting for the unexpected result c. (2 points) Can the orientation of the AraC/GFP insert be determined from these data? If not, why not? If so, what is the insert orientation? d.

Explanation / Answer

1. The band in lane 2 in which no enzyme added near 3000 bp shows the super coiled plasmid DNA.

2. In lane 3 (less visible ) and 4 the visualised band shows linearized DNA.

3. The digestion in 4th lane with Xho I, has no band excised out rather a linear DNA band is visualised.

d. In lane 3 and 5 lane digestion by Sca I, same band pattern is visible in both. If the position of Xho I and Sca I in cloned fragment is already known along with the restrictions sites in vector pGem T then the relative orientation can be determined as the restricted DNA band will be of different lengths in both the orientations.

Since 2 bands are visualised it means the Xho I sites are placed at two corner of insert. Hence according to the position of Xho I on the plasmid vector map, the orientation can be ascertained although Sca I may be found any where either on vector insert.

Vector --------Xho I----------insert --------------Xho I--------- vector

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