What is Multiple Cloning Sites?

A multiple cloning site (MCS) is a section of DNA, usually 20+ restriction sites. These restriction sites are different areas that a gene can be inserted into, and represent complimentary ends of DNA sequences that will adhere to the section of DNA to be inserted.

Restriction sites can have blunt, or 'sticky' ends (like an overhang), and are produced using restriction enzymes that cut the DNA in a specific way. The DNA to be inserted is then cut with the opposing sequence, so that a join can be formed.

MCS are typically found within engineered plasmids (circular double stranded DNA molecules), where they allow sections of inserted DNA be expressed.

An example of this would be the production of Insulin for sufferers of diabetes. The gene for insulin is inserted into a the MCS of a plasmid. The plasmid is then transfected into a bacteria or yeast cell, where the DNA within the plasmid is read, and expressed, leading to the production of insulin.

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Few Questions about Multiple Cloning Sites:

In plasmids such as Bluescript with a multiple cloning site in a modified lacZ gene, a cloned insert can be detected by a change of colour from blue to white?

yes, the insert disrupts the lacZ gene and so if you plate the bacteria with x-gal (a substrate to turn blue), the colonies that have the insert will remain white while the colonies that don't will stay blue. lacZ when uninterrupted allows the x-gal to be metabolized and turn blue.

Why is it important that the multiple cloning site of DNA has only one restriction enzyme recognition site? I know that it makes the plasmid linear instead of circular but why does the plasmid need to be linear?

Because if it has two sites you'll end up with two pieces of DNA instead of one piece.

I have cloned my gene of interest in pUC18 at multiple cloning site. can i use this plasmid for over expression?

Yes, you can use it for over expression. Make sure your plasmid has the proper self cloning sequences, one plasmid per cell won't do you much good.
Precautions? 

Make sure your gene is pure, as in not having anything that might ruin your experiment. Test culture your target bacterium first to weed out the non functionals and then cultivate the good ones, the ones that produce your targe protein. You might need to assay for this stuff though, unless there's one of the specialized agars that will detect your target protein.

Phosphatase treated plasmid, does inhibit cloning?

Phosphatase treatment of a linearised plasmid cut in the multiple cloning site enhances vector circularization, and therefore inhibits cloning of genes into the plasmid during the ligation step.
I think that this is false, but Im not too sure why. Can you help.

it's true because phosphatase prevents vectors from closing in on themselves. This doesn't inhibit cloning but ehances it and allows a greater chance for the insert getting in the plasmid and not just the vector re-closing in on itself.

If you have any other questions about plasmids, cloning in bacteria, etc. feel free to contact me. I do this in lab almost daily

Source(s):

http://en.wikipedia.org/wiki/Multiple_cloning_site
http://www.accessexcellence.org/RC/VL/GG/transfer_and.html
http://www.genomex.com/vector_maps/pBluescript_II_KSplus_map.pdf
http://faculty.plattsburgh.edu/donald.slish/Transformation.html
http://en.wikipedia.org/wiki/Restriction_enzyme