Answer to Question #193576 in Molecular Biology for Daniel

Question #193576
  1. if you want to overexpress a protein in ecoli. The protein is from human ocular cells. What vector would you use to express the protein?. You know the sequence of the gene for the ocular protein, where would you get the DNA you need to clone into the vector? And what regulatory sequences need to be present in your vector for expression to occur?
  2. how will u make a conditional mutant in mice using cre/lox system?

thank you

Expert's answer

1.coli, use a strain supplemented with the appropriate tRNA genes. Solubilize and purify the protein in a well-buffered solution containing an ionic strength equivalent to 300–500 mM of a monovalent salt, such as NaCl. Use immobilized metal affinity chromatography (IMAC) as the initial purification step.

2.The Cre/lox system is one of the most powerful and versatile tools developed for mouse genetics.Cre recombinase removes the stop sequence, so the transgene is expressed only in the cells where Cre is active.

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