hi how can I find useful information about this question or anyone can help me h

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hi

how can I find useful information about this question or anyone can help me how to design a practical?

thanks

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. EcoRI Smal BamHI Write out in brief steps, or via a diagram or both, the strategy / scheme that you would use to carry out this cloning task. In the scheme that you devise, be sure to include any controls; and a simple experimental way of determining that the gene is in the correct orientation (you cannot for instance simply send it out to be sequenced) There is no length limit, but brevity, with completeness, is best. I would recommend something in the range of say one to two pages.

Explanation / Answer

As you have shown that the insert having 3 different restriction sites.

But you are not aware of the orientation of gene of interest in your insert.

In that case, you must double digest your insert and vector with a couple of same restriction enzymes each time in different combinations in separate experiments.

For example, EcoRI and SmaI, EcoR I and BamHI, SmaI and BamH1

It gives the different length of fragments from the insert.

Then, you should ligate insert and vector. But as your interest, you should not go for sequencing to know the right orientation of the insert.

In that case, you have to transform your ligated vector into competent cell like BL21 (E.coli) and then give IPTG induction for overexpressing those genes coded protein

Then, purify that protein through Ni+ column and then run the SDS-PAGE to look at target gene coded protein.

If that expresses, you can see in one your combinations (out of 3) of restriction digestion, those couple of restriction enzymes cleaved fragment would be your interested gene present in your insert. If it is not expressed the orientation of interested gene can be confirmed as inappropriate on your insert.

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