Definition:
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.
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Additional information - Cloning Using PCR, Restriction Enzymes & ligation |
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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