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Maximization of injection volumes for DNA analysis in capillary electrophoresis.
Electrophoresis. 2001 Dec; 22(20):4328-32.E

Abstract

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.

Authors+Show Affiliations

Huang CC
Department of Chemistry, National Taiwan University, Taipei, ROC.
Hsieh MM
No affiliation info available
Chiu TC
No affiliation info available
Lin YC
No affiliation info available
Chang HT
No affiliation info available

MeSH

BuffersDNAElectrophoresis, CapillaryPolymerase Chain Reaction

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11824598

Citation

Huang, C C., et al. "Maximization of Injection Volumes for DNA Analysis in Capillary Electrophoresis." Electrophoresis, vol. 22, no. 20, 2001, pp. 4328-32.
Huang CC, Hsieh MM, Chiu TC, et al. Maximization of injection volumes for DNA analysis in capillary electrophoresis. Electrophoresis. 2001;22(20):4328-32.
Huang, C. C., Hsieh, M. M., Chiu, T. C., Lin, Y. C., & Chang, H. T. (2001). Maximization of injection volumes for DNA analysis in capillary electrophoresis. Electrophoresis, 22(20), 4328-32.
Huang CC, et al. Maximization of Injection Volumes for DNA Analysis in Capillary Electrophoresis. Electrophoresis. 2001;22(20):4328-32. PubMed PMID: 11824598.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Maximization of injection volumes for DNA analysis in capillary electrophoresis. AU - Huang,C C, AU - Hsieh,M M, AU - Chiu,T C, AU - Lin,Y C, AU - Chang,H T, PY - 2002/2/5/pubmed PY - 2002/7/12/medline PY - 2002/2/5/entrez SP - 4328 EP - 32 JF - Electrophoresis JO - Electrophoresis VL - 22 IS - 20 N2 - We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/11824598/Maximization_of_injection_volumes_for_DNA_analysis_in_capillary_electrophoresis_ L2 - https://doi.org/10.1002/1522-2683(200112)22:20<4328::AID-ELPS4328>3.0.CO;2-H DB - PRIME DP - Unbound Medicine ER -