Our assignment is to give protocols on how we would produce aprotein by cloning

Question

Our assignment is to give protocols on how we would produce aprotein by cloning a gene coding for that protein. I've lookedeverywhere and I can't figure out what to do here, mainly becausethe question indicates that whatever procedure needed must be donebefore ligation:

There is no Blue/White Screen using pET28b+, so what must you dobefore ligation? What is the protocol for thisprocedure?

Here is a PDF of pET28b(+):www.merck-chemicals.com/showBrochure?id=200905.345
A map on EcoliWiki:http://ecoliwiki.net/colipedia/index.php/pET-28a(%2B)

Thanks for your help.

Explanation / Answer

blue/white screening allows you to assess whether or not yourdesired insert is in the correct orientation. so the question ispointing to something that can cause improper ligation which couldbe determined normally with blue/white screening. before you ligatethe vector with the insert, the vector must be digested and containcompatible ends with the digested insert. depending on therestriction enzymes you use, the vector can be digested in a waythat creates cohesive compatible ends. in the case of this, youwould need to treat the vector with a phosphatase. doing so willprevent the vector from recircularizing with itself and containingnone of your desired insert.

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