genetics pse

Arabidopsis thaliana has among the smallest genomes in higher plants, with a haploid genome size of about 100 Mb. If this genome is digested with BbvCl, a restriction enzyme which cuts at the sequence CCTCAGC GGAGTCG approximately how many DNA fragments would be produced? Assume the DNA has a random sequence with equal amounts of each base. 100,000,000 bp/ 47 = 61 A newly discovered insect has a genomic DNA that does not contain any repetitive DNA or any intronic regions. The insect’s genomic DNA has been treated with the restriction enzyme EcoRI. One of the fragments (with a length of 4 kb) produced in this manner is isolated and ligated into a pUC19 plasmid vector that has also been treated with EcoRI. This recombinant construct is called plasmid A. Samples of plasmid A, pUC 19, and the total genomic DNA from the insect are digested completely with the restriction enzyme EcoRI. The three digests are loaded into separate wells: plasmid A (lane 1), pUC19 (lane2) and total insect genomic DNA (lane 3) of an agarose gel and subjected to electrophoresis. Draw a gel (you can do it by hand) showing what will be seen in the lanes 1, 2, and 3 of the gel after it is stained with ethidium bromide for visualizing DNA. (indicate molecular sizes where appropriate) Draw what will be seen in each lane if the gel is transferred to a membrane and subjected to Southern blotting and the 4000 base pair genomic DNA fragment (labeled with a fluorescent tag) from the insect is used to probe the blot. Your PhD thesis advisor has given you the task of preparing a human genomic DNA library. a. How will you prepare DNA fragments from the human genomic DNA for use in construction of this library assuming that you want the insert sizes to be about 20000bp in size? To prepare DNA fragments from the human genomic DNA I would use a restriction enzyme to “cut up” the DNA into fragments. This would produce fragments of different size that are suitable for packaging. b. What vector will you use for construction of this library? A Lamda vector is normally used to construct a genomic library. c. How will you ligate the genomic DNA fragments into the vector? In order to ligate the genomic DNA fragments into the vector I would use the Lamda phage DNA as well as the DNA I am attempting to insert into the vector. Once the restriction enzyme cleaves them into fragments the phage arms, which contain all the genes needed for replication are broken apart and the insert goes in between the left and right arm of the phage. They are then ligated together with enzymes such as ligase and this creates recombinant DNA. d. What host will you use for propagation of your library? I would most likely use a bacteria cell as a host for the propagation of the genomic library. f. What probe would you use for screening your library assuming that you wanted to isolate the human insulin gene? Knowing that insulin is produced in the pancreas, I would use a probe that contains pancreatic cells. e. How many clones would you have to screen to be pretty sure that you will indeed find a clone for insulin? 30,000, because there are about 10,000 variants of each gene and it is safe to repeat 3 times to ensure you will find the correct clone.