Question

(1) You start with the pUC18 plasmid that is a -2.7 kb circular DNA molecule. The multiple cloning site (i.e., polylynker) has unique restriction sites in the following order: HindIII, BamHI, EcoRI. Recall that unique restriction site means that these sites are only found once on the pUC18 plasmid. The pUC18 plasmid is digested with BamHI and EcoRI, and a 5 kb insert of stingray DNA with BamHI and EcoRI sticky ends (i.e., cohesive ends) is ligated to form a -7.7 kb circle. The circle is subject to single and double digests, and the resulting fragments in kb) are shown below. Please draw a map of the 7.7 kb circular recombinant DNA molecule, with the restriction sites. E/H E/B BamHI (B) 5.4 HindiII (H) 4.7 3.0 EcoRI (E) 4.7 3.0 23 3.0 2.4 2.3 4.4 2.7 0.3 (thick) 2.7 (thick) 2.0 0.3 (2) A 10 kbp linear DNA fragment is digested with Pvull, EcoRV, or Pvull and EcoRV. Prior to digestion, the ends of the DNA molecule are labeled with radioactive phosphorus. After digestion, DNA is subject to gel electrophoresis, EtBr staining, gel photography, and then to autoradiography. In this way, all bands are recorded, and those that are radioactive (*) are noted as well. The data are: 4.7* 3.0* Pvull EcoRV Pvull/EcoRV 4.0 3.0* 3.1 2.8 2.5* 2.2* 2.2* 0.5 1.2 0.5 0.3 *Radioactively labeled fragments Draw the linear DNA restriction map with Pvull and EcoRV sites.

Can someone please help me answer these? Neat work is greatly appreciated.

Explanation will also be appreciated (optional, if you have time).

Thank you so much! I will give a big thumbs up :)

Answer 1

Answer-1

The map of 7.7kb recombinant plasmid will be as following figure.The position of restriction enzymes EcoRI, BamHI and HindIII are represented.

5kb insert Hindili 0.3kb ECORI 2kb EcoRI BamHI 0.3kb Vector backbone BamHI Hindill 2.4kb