You have cloned a Pvull fragment 3.997kb in length, and have ligated this fragme

Question

You have cloned a Pvull fragment 3.997kb in length, and have ligated this fragment into the vector pQE16(3.768kb in length), creating a new plasmid. However, the orientation of the Pvull fragment in the vector is not known. You know that there is a single BgIII site in the pQE16 vector, and that there is a single BgIII site in the Pvull fragment. Based on the gels below, diagram a circular plasmit showing the orientation of the Pvull fragment.

My initial problem is that I'm finding it hard to determine the size of the fragments

Explanation / Answer

So you should create a standard curve. Measure the scale given. Measure from the origin down to each size point given and then graph distance traveled on the x axis and known size on the y axis. This should be done on a log scale and a best fit line added. You can do this in Excel quite easily. Then once you have this standard curve you measure the distance each of the digested fragments migrated and use that distance to find the size on the standard curve you made. If you use excel and add a best fit line then you can get the line equation and just measure each digested fragments distance and plug these in for x and solve the equation for y to get the fragment size of the band. That should get you all the band sizes.

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