You cloned your favorite gene, TGIF, into a circular bacterial plasmid vector pN

Question

You cloned your favorite gene, TGIF, into a circular bacterial plasmid vector pNIM. You identify two recombinants, plasmid X and plasmid Y, that contain the TGIF gene inserted in pNIM. You digest all three plasmids (pNIM, plasmid X, and plasmid Y) with EcoRI, BamHI, or both restriction enzymes together. All digestions were complete and gave the following results:

(a) On the templates provided, draw possible restriction maps of plasmids X and Y which are consistent with the results presented. Your maps must indicate which restriction sites are in the vector (thin line) and which restriction sites are in the insert (thicker line). Include the relative distances between the restriction sites. Below the templates, indicate the size of the insert and the insertion site (the vector’s restriction site in which the insert was introduced).

(b) A linear DNA fragment was digested with enzymes A, B, and C. The following table indicates the fragment sizes obtained from complete digestions.

Draw a restriction map on the template provided which is consistent with the data obtained. Indicate the relative distance between each of the restriction sites. Note that the position of restriction site “A” is indicated.

ANSWER:

show me how to do it :)

Explanation / Answer

Hi,
The restriction maps are generated by calculating and appropriately organizing the band sizes. In the given figure, consider the Plasmid X map ( the one of left). If you look at the table above, the EcoR1 cuts at three places and produces bands of 9, 4, 3 size. So, do the first cut on the plasmid anywhere you like. Then is the second cut 4kb apart, and then is cut 9kb apart from second cut. The size of cut 1 and cut 3 will be 3 kb. Similarly, do for HindIII. IF we see only BamHi we will not get much information. But when both enzymes cut the plasmids,we get 5, 4, 3, 2 kb fragments. the fragment of 4 and 3 were also formed in EcoRi alone reaction. None of the fragments of BamHI alone reaction are found in combined enzyme reaction. So the EoR restriction sites are within the BamHI. SO we get the first cut of Bam between Eco 1st and 2nd cut,to get 2kb size fragments. then another bam cuts at 4kb from eco 2nd cut generating a fragment of 4 and 5kb. Thus by analyzing the restriction fragments we can guess the restriction map of plasmids.

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