Operational variables in high-performance gel filtration of DNA fragments and RNAs

Double-stranded DNA fragments and ribosomal and transfer RNAs were measured by high-performance gel filtration on TSK-GEL G2000SW, G3000SW, G4000SW and G5000PW columns to investigate the separation range and resolution of these columns and the effects of eluent ionic strength and flow-rate on retention and resolution. These columns could separate double-stranded DNA fragments up to ca 1 X 10(6) and rRNAs up to ca 5 X 10(6) daltons in molecular weight. However, it was found that the selection of the column is very important to achieve optimum separation, depending on the molecular weight of the sample. Elution is delayed as the eluent ionic strength is increased. An eluent ionic strength of 0.3-0.5 seemed appropriate in most cases. Resolution is greatly increased as the flow-rate is decreased.