MCS (Multiple Cloning Site) / Polylinker

This region contains several restriction enzyme recognition sites very close to each other. Each of these sites cuts the plasmid only once. Hence, cutting the plasmid with one of these restriction enzymes does not disrupt any of its essential features, and may serve to  insert the foreign DNA. By having many different restriction enzyme recognition sites close together, polylinkers offer many choices in regard to which enzymes to use for cloning.

Experimental Tips

Additional information - Cloning Using PCR, Restriction Enzymes & ligation

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Experimental Tips:

Blue/White screening: Some vectors contain the MCS within the Lac Z gene, so that successful transformations result in white colonies, whereas plasmids without the insert give rise to blue colonies. Using the Blue/White screening method ensures that the target insert has been successfully cloned. For more details, see Promega's publication .
In addition to blue/white screening, recombinant colonies may be screened for the presence of the desired insert by restriction digestion of miniprep plasmid DNA or by direct PCR amplification of bacterial colonies.

TA cloning: this method is relevant for cloning of PCR products. Some polymerases add a single deoxyadenosine (A), in a template -independent fashion, to the 3´-ends of the amplified fragments. This makes it possible to clone the resulting PCR products directly into a linearized cloning vector with single 3'-T overhangs. For more details, see the  pGEM Easy Vector manual.

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