You work in the lab that studies enzyme kinetics. Part of your project is to clo

Question

You work in the lab that studies enzyme kinetics. Part of your project is to clone a gene of interest in a bacterial system using a vector with inducible promoter to test the enzyme activity under various hypoxic conditions. You can afford to buy a vector that has a MCS with two RE blunt ends utilizing the REs routinely used in your lab. However, upon investigating the vector map, you notice that the two restriction sites are 86 bps away. Do you buy the vector for your experiment (this is the only vector that fits your budget at this point)? If so, what do you anticipate consequently, and what (if anything) do you propose to implement/modify in your experimental design to successfully express the enzyme of interest?

Explanation / Answer

Let us identify the problem here first. When you cleave the vector using RE and add ligase post addition of the gene of interest chances are that the vector might self-ligate and your gene would not be added to the vector. SO we add linker DNA, Now we use the RE to get sticky ends. Now upon ligation, the vector and gene of interest will have more chances of ligating to themselves.

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