Name BIOL-3311 Molecular Biology Assignment In order to assay and cut the ID enz

Question

Name BIOL-3311 Molecular Biology Assignment In order to assay and cut the ID enzymes. the products of your cloning reaction you prepare DNA from several bacterial colonies When you run these reactions on an agarose gel you see the following banding pattern: Lanel DNA size marker Lane 2 PCR product undigested 500 Lane 3-PCR product digested 400_ with HinD3 300 Lane 4- PCR product digested with Bamtn 100_ 4) Below there is a line that represents the PCR product. The small vertical lines on this product represent the 100, 200, and 300 bp marks along its length. Please indicate the approximate location of the HinD3 and BamHl sites on this line with an H and a B respectively. (4 pts) Note that two scenarios are possible. You need only illustrate one correct possibility) 100 200 300 400

Explanation / Answer

Question 1

a) The ~400bp product most likely encodes the gene.

b) The gene is known to produce a protein product that is ~133 amino acids long. Each amino acis is encoded by 3 nucleotides (bases). Therefore 133 amio acids are encoded by 133 x 3 = 409 nucleotides or bases. The two products from PCR are ~400 bp and ~1300 bp long. Both of them are assumed to contain only exons and no introns. Hence, only coding nucleotides are present in both. Therefore clearly, the product that codes for the gene of interest (~133 aa long) is the ~400 bp one.

Question 2

The restriction enzyme (in this case, EcoRI) cuts the DNA assymetrically, causing the formation of 5' overhangs both on the plasmid and on the DNA insert. These overhangs are cut with the same restriction enzyme sites, which means those cuts sequences were similar before they were cut assymetrically. Now the newly generated 5' overhang of plamid is complimentary to the sticky end of the DNA insert on one assymetrically cut end, and the 5' overhang of the DNA insert in the other end is complimentary to the sticky end of plasmid. This causes the perfect ligation of these 5' overhangs and complimentary sticky ends, resulting in the perfect incorporation of DNA insert into the plasmid.

This sticky end joining property is essentially due to the complimentarity of nucleotides or bases.

Question 3

DNA ligase enzyme. This causes the complimentary pieces of DNA to ligate permanantly.

Question 4

                 0-------1---------2--------3-------4

                 I--------B--------H---------I--------I

0--> 0,   1--> 100bp,   2--> 200 bp,   3--> 300bp,   4--> 400bp

Answer is:    0--------B--------H--------3--------4

The H marker is at 200 bp (to generate two fragments each 200bp in length; 0--------200 and 200------400)

The B marker is either at 100 bp or at 300 bp (to generate one fragment that is 100bp long and another 300 bp long; 0-----100 and 100---------------400 or, 0-----------------300 and 300-----400)

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