Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q

Anal Biochem. 1987 Oct;166(1):158-71. doi: 10.1016/0003-2697(87)90558-6.

Abstract

Separation of DNA restriction fragments by FPLC ion-exchange chromatography on Mono Q and Mono P columns was investigated. The columns were found to be particularly suitable for the separation of fragments up to 500-600 bp long. Larger fragments can also be separated although less effectively. We found the following practical working ranges for the parameters investigated: pH, 4 to 11; flow rate, 0.05 to 0.6 ml/min corresponding to separation times between 2 and 20 h. (better resolution is achieved at lower flow rates); gradient slope; between 0.5 mM eluting salt/ml buffer and over 5 mM/ml (better resolution is achieved at lower gradient slopes; eluting ionic strength was found to be independent of gradient slope); gradient composition, chloride salts of smaller monovalent cations eluted the DNA at lower ionic strengths but separations obtained were similar; additives, substances such as urea, formamide, and EDTA can be added without chromatographic effects; sample amount: amounts from 2.5 to 200 micrograms were applied, corresponding to single peak content of from 42 ng to 74 micrograms DNA. Yields were generally over 90% and the chromatographed DNA was fully accessible to restriction enzyme cleavage. Separations occurred predominantly according to DNA size, but AT-rich fragments were retarded in a predictable way.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange / methods
  • DNA / isolation & purification*
  • DNA Restriction Enzymes
  • Hydrogen-Ion Concentration
  • Indicator Dilution Techniques
  • Particle Size
  • Polymorphism, Restriction Fragment Length

Substances

  • DNA
  • DNA Restriction Enzymes