?C mutation was synthesized as both wild-type (SV/luc/CDK6wt) and mutant (SV/luc/CDK6mut) versions using GeneArt Strings (Life Technologies, Mulgrave, VIC, Australia) and cloned into the downstream multiple cloning site of pGL2P (Promega, Alexandria, NSW, Australia).
Most also contain a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
The carefully chosen enzyme cuts the plasmid in the Multiple Cloning Site only once. The Multiple Cloning Site is situated in the middle of the Lac gene, which codes for the galactosidase.
2) The Insert is cut with the same enzyme (s) as the vector ...
Vector-specific PCR primers flanking the multiple cloning site (MCS) and containing either T7 or SP6 RNA polymerase promoters were used to synthesize double-stranded DNA templates for the labeling in vitro transcription reaction.
Cloning vectors minimally contain an origin of replication, selectable marker gene (e.g., ampicillin resistance gene), and multiple cloning site containing unique restriction enzyme sites; other useful features may also be present.
Cloning siteA restriction enzyme site or group of sites (= ~) located in a vector at the best position for insertion of DNA.
Colony liftBlot taken from colonies (normally bacterial) growing on an agar plate.
See also: What is the meaning of Trans, Cloning, DNA, Vector, Sequence?