11. You are pleased because you think you\'ve obtained a positive clone of pGEM-
ID: 214367 • Letter: 1
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 end of the exam. DigestiResults? 1. Laddert 2. Notenzyme 3. Scal 4. Xhol 5. Scal/Xhol (2 points) Circle the band(s), if any, that represents supercoiled plasmid DNA. a. 8000 6000 5500 4000 (2 points) Box the band(s), if any, that represents linearized plasmid DNA. b. 3000 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
Ans
a.there is no supercoiled plasmid DNA bacausesupercoiled DNA appears as thick band.
b lane no 4 shows linear plasmid DNA.
c.Lane 3 is unexpected because looking at the plasmid files below there is only one site for ScaI enzyme which should show linear single band but the lane is showing 2 bands.
d. yes we can determine the orientation of the plasmid.Now according to the plasmid files given below when we observe the reverse plasmid at the bottom, Xhol site is at 436 bp and Sca I site is at 4018 bp.So when these two restriction enzymes are used then we will get first nick at 436 bp and then at 4018 bp.The one band will be of 3582(4018-436) bp and next will be of 1561(5143-3582) bp.which is not seen in lane 5.If we observe forward plasmid vector first nick is at 1809bp and then at 4018bp so the bands will be of 2209bp(4018-1809) and 2934(5143-2209). Which is seen in lane 5.So the orientation is in forward direction.
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