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Molecular Cloning

pBR322 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 DNA sequence, sequence analysis and map creation see free on-line tool REviewer™.

Applications

• Cloning.
• Preparation of DNA molecular weight standards.

Description

pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in length and contains:
1 –  the replicon rep responsible for the replication of plasmid (source – plasmid pMB1);
2 – the rop gene, coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1);
3 – the bla gene, coding for beta-lactamase, that confers resistance to ampicillin (source – transposon Tn3);
4 – the tet gene, encoding tetracycline resistance protein (source – plasmid pSC101).

The circular sequence is numbered such that 1 is the first T of the unique EcoRI site GAATTC and numbering increases through the tet gene, the pMB1 material and finally through the Tn3 region.

The map shows enzymes that cut pBR322 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 4153-4085 (complementary strand) code for a signal peptide. The indicated rep region is sufficient to promote replication. DNA replication initiates at position 2533 (+/- 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.

GenBank/EMBL Accession Numbers
J01749, K00005, L08654, M10282, M10283, M10286, M10356, M10784, M10785, M10786, M33694, V01119.

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 AluI, BsuRI 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. Bolivar, F., et al., Construction and characterization of new cloning vehicles. II. A multipurpose cloning system, Gene, 2, 95-113, 1977.
2. Covarrubias, L., et al., Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328, Gene, 13, 25-35, 1981.
3. Peden, K.W., Revised sequence of the tetracycline-resistance gene of pBR322, Gene, 22, 277-280, 1983.
4. Sutcliffe, J.G., Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322, Proc. Natl. Acad. Sci. U.S.A., 75, 3737-3741, 1978.
5. Sutcliffe, J.G., Complete nucleotide sequence of the Escherichia coli plasmid pBR322, Cold Spring Harb. Symp. Quant. Biol., 43, Pt 1, 77-90,1979.
6. Watson, N., A new revision of the sequence of plasmid pBR322, Gene, 70, 399-403, 1988.