High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

Journal Article · Tue Dec 27 00:00:00 EST 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00426a019· OSTI ID:5518536

Mathew, M K; Smith, C L; Cantor, C R ^[1]

---

1. Columbia Univ., New York, NY (USA)

Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1,500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.

• OSTI ID: 5518536
• Journal Information: Biochemistry; (USA), Vol. 27:26; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
CALIBRATION STANDARDS
ELECTROPHORESIS
TEMPERATURE EFFECTS
BACTERIOPHAGES
ELECTRIC FIELDS
MASS TRANSFER
POLYSACCHARIDES
SACCHAROMYCES CEREVISIAE
SIZE
CARBOHYDRATES
EUMYCOTA
FUNGI
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PLANTS
SACCHARIDES
SACCHAROMYCES
STANDARDS
VIRUSES
YEASTS
550200* - Biochemistry