pUC18
pUC18 and pUC19 vectors are small, high copy number, E.coli
plasmids,
2686 bp in length. They are identical except that they contain
multiple cloning sites
(MCS) arranged in opposite orientations. pUC18/19 plasmids contain:
(1) the pMB1 replicon rep
responsible for the replication of plasmid (source – plasmid pBR322).
The high copy
number of pUC plasmids is a result of the lack of the rop gene
and a single point
mutation in rep of pMB1; (2) bla gene, coding for
beta-lactamase that
confers resistance to ampicillin (source – plasmid pBR322). It differs
from that of
pBR322 by two point mutations; (3) region of E.coli operon lac
containing
CAP protein binding site, promoter Plac, lac repressor binding
site and
5’-terminal part of the lacZ gene encoding the N-terminal
fragment of
beta-galactosidase (source – M13mp18/19). This fragment, whose
synthesis can be
induced by IPTG, is capable of intra-allelic complementation with a
defective form of
beta-galactosidase encoded by host (mutation lacZDM15). In the
presence of IPTG,
bacteria synthesise both fragments of the enzyme and form blue
colonies on media with
X-gal. Insertion of DNA into the MCS located within the lacZ
gene (codons 6-7 of lacZ
are replaced by MCS) inactivates the N-terminal fragment of
beta-galactosidase and
abolishes alfa-complementation. Bacteria carrying recombinant plasmids
therefore give rise
to white colonies.
pCMV-b-gal
This is a high copy
number eukaryotic vector, pCMVb expresses the full-length b-galactosidase gene under the control of the
cytomegalovirus immediate early gene (CMV IE) promoter. This
vector is very useful for transfection
of mammalian cells in culture and for use in other species. The b-galactosidase enzyme expression
is enhanced
by elements including: SD/SA-RNA splice donor and acceptor sequence, and
SV40
late polyadenylylation signal. pCMVb expression vector also contains b-lactamase gene, which acts as a
selection marker (100mg/mL ampicillin resistance) in E.
coli
host. pCMVb vector has been
tested to generate up to
2530u/mg cell extract (MacGregor, and Caskey).
In addition, the b-galactosidase gene can be
excised using the
NotI sites to allow the insertion of other genes to be expressed
under
the same regulatory elements in mammalian cells.
pIRES2-EGFP
pIRES2-EGFP replaces (but is not derived from) the pIRES-EGFP Vector previously sold by BD Biosciences Clontech. pIRES2-EGFP is functionally similarly to pIRES-EGFP; however, pIRES2- EGFP gives brighter EGFP fluorescence than the older vector.
No comments:
Post a Comment