doi:10.1016/j.chroma.2006.11.027 
Copyright © 2006 Elsevier B.V. All rights reserved.
Effect of salt on purification of plasmid DNA using size-exclusion chromatography
Liang-Zhu Lia, b, Yong Liub, Mao-Sheng Suna and Yi-Ming Shaob, 
, 
aInstitute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, China
bNational Center for AIDS/STD Prevention and Control, China CDC, Beijing, China
Received 9 July 2006;
revised 22 October 2006;
accepted 8 November 2006.
Available online 21 November 2006.
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Abstract
In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5 M, 1 M, 2 M, respectively) of two types of salt (NaCl and (NH4)2SO4) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.
Keywords: Size-exclusion chromatography; High salt; Genomic DNA; Supercoiled plasmid DNA; RNA; Size-exclusion mode; Interaction mode
Fig. 1. Curves of size-exclusion chromatography. (a) TE buffer. (b, c, d) three concentrations of sodium chloride in the buffers. (b: 0.5 M; c:1 M; d: 2 M). (e, f, g) three concentrations of ammonium sulfate in the buffers(e: 0.5 M; f: 1 M; g: 2 M). The first peak is pDNA (shown by arrow), and the second peak is RNA mainly together with some other small contaminating molecules. (dashed line = conductivity profile).
Fig. 2. Curves of E.coli genomic DNA (gDNA) in the size-exclusion chromatography. (a) TE buffer. (b, c, d) three concentrations of sodium chloride in the buffers (0.5, 1 and 2 M, respectively). (e, f, g) three concentrations of ammonium sulfate in the buffers (0.5, 1 and 2 M, respectively). X-axis represents the fractions (the same number fraction was corresponding to the same retention volume). Y-axis represents the residual amount of gDNA (ng μl−1).
Fig. 3. The average amount of residual E. coli genomic DNA (gDNA) after gel filtration in different buffers. From TE to 2 M AS, the average amount of residual E. coli gDNA is 4.48, 2.3, 2.26, 1.71, 0.8, 0.69 and 0.08 ng μl−1, respectively. (The average amount of plasmid is about 250 ng μl−1.)
Fig. 4. Comparison of the effects of 2 M AS and 2 M NaCl by Southern-blotting analysis. (A) Negative control: DNA dilution buffer (5 μg Herring Sperm DNA per 100 μl TE buffer). (B) Standard sample 1: 10 ng gDNA per 100 μl TE buffer. (C) Standard sample 2: 100 ng gDNA per 100 μl TE buffer. (D) Sample 1: purified by gel filtration in presence of 2 M AS. (E) Sample 2: purified by gel filtration in presence of 2 M NaCl. (F) Sample 3: no-purified by gel filtration.
Fig. 5. Agarose gel electrophoresis map for samples which were collected by fraction collector. OC: open circular; SC: supercoiled plasmid. The order of fractions is the same as in Fig. 2.
Fig. 6. The proportion of supercoiled plasmid DNA in different conditions (scanned by Quantity One software (Bio-Rad)). X-axis represents the fractions which were collected in each process. Same number fraction was collected at same retention volume. Y-axis represents the proportion of sc plasmid DNA.
Fig. 7. Analytical IEX on Mono Q FPLC. Plasmid DNA, which was pre-purified by SEC on Sephacryl S-1000 SF, was analyzed on a 10 μm porous anion exchanger (MonoQ FPLC), using a linear NaCl gradient ranging from 0 to 2 M NaCl. (a) There is no salt in the running buffer. (b, d, f) three concentrations of sodium chloride in the buffers (0.5, 1 and 2 M, respectively). (c, e, g) three concentrations of ammonium sulfate in the buffers(0.5, 1 and 2 M, respectively). In graphs, peak 1 has a strong absorbance at 215 nm, so we concluded it is saccharide; peak 2, peak 3 and peak 4 was identified by gel electrophoresis (as shown in graph h): the peak 2 (lane 2) is linear DNA; the peak 3 (lane 3) is supercoiled DNA (shown by arrow) and the peak 4 (lane 4) includes gDNA and open circular DNA. (dashed line = conductivity profile).
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