Sequential elution of denatured proteins, hydrolyzed RNA, and plasmid DNA of bacterial lysates adsorbed onto stacked DEAE-cellulose membranes.
Modified membrane chromatography is emerging as a widely applicable technique for the separation of macromolecules. The use of DEAE-cellulose membranes (MemSep) for the purification of bacterial plasmid DNA has been assessed. Cleared bacterial lysates were injected directly onto the membranes without any prior sample cleanup. A single phenol extraction of adsorbed DNA was carried out and about 60 times less phenol was required to achieve the same extent of deproteination for DNA adsorbed onto the membranes as compared to DNA in solution. After chloroform and ethanol wash, (oligo)ribonucleotides resulting from RNase treatment were desorbed with 0.3 M LiCl, 5 mM LiOH. Finally, DNA was eluted with 0.5 M NaCl, 5 mM NaOH. The complete elution of DNA required NaOH in addition to NaCl and the latter salt was a better eluent than LiCl. Without RNase treatment, plasmid DNA-RNA complex required 2 M NaCl, 5 mM NaOH to be completely desorbed. The whole procedure took less than 40 min. The DEAE-cellulose membranes can withstand more than 100 cycles of regenerations and uses without any noticeable decrease of their binding capacity. No cross-contamination of successive DNA preparations was observed. Plasmid DNA was a good substrate for DNA endonucleases. Restriction fragments repurified by this procedure were amenable to ligation. Transformation of Escherichia coli and Saccharomyces cerevisiae with plasmid DNA was observed.
van Huynh
N
N
Ministère de l'Agriculture, Institut de Recherches Chimiques, Tervuren, Belgium.
Motte
J C
JC
Pilette
J F
JF
Decleire
M
M
Colson
C
C
D000327:Adsorption; D001426:Bacterial Proteins / Q000302:isolation & purification*; D003636:DEAE-Cellulose; D006868:Hydrolysis; D008567:Membranes, Artificial; D009691:Nucleic Acid Denaturation; D010957:Plasmids / Q000302:isolation & purification*; D011489:Protein Denaturation; D012329:RNA, Bacterial / Q000302:isolation & purification*
D001426:Bacterial Proteins; D008567:Membranes, Artificial; D012329:RNA, Bacterial; D003636:DEAE-Cellulose