The question is DESIGN AN EXPERIMENT FROM THE DETAILS BELOW You need to perform

Question

The question is DESIGN AN EXPERIMENT FROM THE DETAILS BELOW You need to perform an experiment that will result in the cloning of a gene into the expression vector pUC18. A map of pUC18 with all features indicated, including the multi-cloning site, may be easily obtained by typing the vector name into Google. The insert containing the gene is a blunt-ended fragment that was not generated with a restriction enzyme. The name and size of the gene is irrelevant. We are interested only in the cloning strategy. However, the insert is 2.4 kb, a map of which is shown below with unique restriction sites indicated. And i found the answer ( It was mentioned that the gene is a part of the larger 2.4 kb insert (and the sequence is not at all irrelevant in this case). As you are going to express it into pUC18, thyen I assume that the sequence is known to you. Design primers and append restriction sites to them of your favourite restriction enzymes, based on the pUC18 cloning site. Amplify it, digest both the amplicon and the vector, then ligate. Transform to competent E. coli along with the intact pUC18 as a control. If the sequence is unknown, you only have to go for blunt end ligation and one more extra cloning step. Blunt end cloning is notoriously difficult. the CloneJet PCR-cloning kit (Thermo) can be used and the best method for this purpose. Get the clone, amplify with primers designed for the flanking regions of the insert ends (vector ends) with pUC18 restriction site appended, clone into pUC18) . just i want more details about the experiment such as Methodology: (design of experimental scheme, controls, standards, as required)

Explanation / Answer

The pUC18 is a circular double stranded (ds) DNA molecules. The pUC18 is a plasmid that is genetically engineered to include antibiotic resistance and gene, which encodes enzyme beta-galactosidase.

The thermostable DNA polymerase is used to amplify the vector and amplicon. In this method both blunt-end and sticky-end DNA fragments can be cloned. The process of ligation takes place for 5 minutes. Before ligation the fragments are subjected to blunting for 5 minutes in presence of thermostable DNA blunting enzyme. The transformed vectors are inserted into the suitable host (E.coli).

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