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Vectors and Phage DNA

pUC18, pUC19 DNA

Product information

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Features

• Purified by chromatography using proprietary patented technology.
• More than 90% in the supercoiled form.
• Isolated from E.coli (dam+, dcm+).
• For pUC18 DNA sequence, pUC19 DNA sequence, sequence analysis and map creation see free on-line tool REviewer™.

Applications

• Cloning.
• Sequencing of insert DNA.
• pUC18 DNA: Preparation of DNA molecular weight standards.

Description

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 the replicon rep of pMB1;
2 – the 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 – the region of E.coli lac operon containing a CAP protein binding site, promoter P_lac, lac repressor binding site and the 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 (alpha) complementation with a defective form of beta-galactosidase encoded by the host (mutation delta(lacZ)M15). In the presence of IPTG, bacteria synthesize 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 alpha-complementation. Bacteria carrying recombinant plasmids therefore give rise to white colonies.

The map shows enzymes that cut pUC18/19 DNA once. Enzymes produced by Fermentas are shown in orange. The coordinates refer to the position of the first nucleotide in each recognition sequence.

The exact positions of the genetic elements are shown on the map (termination codons included). The bla gene nucleotides 2486-2418 (complementary strand) code for a signal peptide. The LacZ polypeptide corresponding to wt beta-galactosidase and essential for blue/white screening ends at nt position 236 (compl. strand); another 30 codons in the same reading frame are derived from pBR322. The indicated rep region is sufficient to promote replication. DNA replication initiates at position 866 (+/- 1) and proceeds in the direction indicated. Plasmids carrying the pMB1 and ColE1 replicons are incompatible, but they are fully compatible with those carrying the p15A replicon (pACYC177, pACYC184). pMB1-derived plasmids can be amplified using chloramphenicol.

Additional Information

• CAP protein binding site – 591-554 (compl. strand);
• mRNA (LacZ) starts at nt position 507 (compl. strand);
• lac repressor binding site – 507-487 (compl. strand).

GenBank/EMBL Accession Numbers
For pUC18 – L09136;
for pUC19 – L09137.

Storage Buffer

10 mM Tris-HCl (pH 7.6) and 1 mM EDTA.

Quality Control

DNA concentration is confirmed spectrophotometrically. Identity and homogenity of DNA is confirmed by digestion by NdeI+HindIII and HindIII, and analysis of DNA fragmentation patterns.

REviewer™

Use for DNA sequence analysis and map creation.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

References

1. Yanisch-Perron, C., et al., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, Gene, 33, 103-119, 1985.