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Research Journal of Microbiology
Jebakumar Solomon, R.D., Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India
Satheeja Santhi, V., Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India
The aim of present study was to compare the biological activity of plasmid purified by CsCl2 buoyant density gradient centrifugation and anion exchange HPLC elution from alkaline cell lysates. The recombinant plasmids were purified from E. coli clones by anion exchange HPLC and CsCl2 density gradient centrifugation. Purified samples were digested with restriction endonuclease and the ligated recombinant plasmids were used to transform E. coli HB101 strain. The CsCl2 purified plasmid DNA exhibited higher degree of purity than HPLC purified plasmid. However, transformation efficiency was higher for HPLC purified samples; recombinant pUC8 in VE144 varied between 2×103 to 3.5×103 for crude sample, 2.52×103 to 4.2×103 for CsCl2 density gradient purified sample and 7.5×104 to 1.15×105 transformants/μg of HPLC purified sample. The pBR322 in VE145 also showed transformation efficiency as 3.9×10 3 to 1.2×104 for crude sample, 5.6×10 4 to 6.3×105 transformants/μg of CsCl2 purified and 4.5×105 to 5.04×106 transformants/μg of HPLC purified plasmid. Due to the higher transformation efficiency and less time consumption, plasmid purification by HPLC can imply major impact on recombinant DNA technology. © 2007 Academic Journals.
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Evaluation of transformation ability of caesium chloride density gradient centrifugation and ion exchange high performance liquid chromatography purified plasmids
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Jebakumar Solomon, R.D., Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India
Satheeja Santhi, V., Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India
Evaluation of transformation ability of caesium chloride density gradient centrifugation and ion exchange high performance liquid chromatography purified plasmids
The aim of present study was to compare the biological activity of plasmid purified by CsCl2 buoyant density gradient centrifugation and anion exchange HPLC elution from alkaline cell lysates. The recombinant plasmids were purified from E. coli clones by anion exchange HPLC and CsCl2 density gradient centrifugation. Purified samples were digested with restriction endonuclease and the ligated recombinant plasmids were used to transform E. coli HB101 strain. The CsCl2 purified plasmid DNA exhibited higher degree of purity than HPLC purified plasmid. However, transformation efficiency was higher for HPLC purified samples; recombinant pUC8 in VE144 varied between 2×103 to 3.5×103 for crude sample, 2.52×103 to 4.2×103 for CsCl2 density gradient purified sample and 7.5×104 to 1.15×105 transformants/μg of HPLC purified sample. The pBR322 in VE145 also showed transformation efficiency as 3.9×10 3 to 1.2×104 for crude sample, 5.6×10 4 to 6.3×105 transformants/μg of CsCl2 purified and 4.5×105 to 5.04×106 transformants/μg of HPLC purified plasmid. Due to the higher transformation efficiency and less time consumption, plasmid purification by HPLC can imply major impact on recombinant DNA technology. © 2007 Academic Journals.
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