Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads(SSMBs)

Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Review of micro/nano technologies and theories for electroporation of biological cells

Electroporation (EP) is one of the important techniques for the introduction of genes and drugs into cells with intense pulsed electric field to induce nanometer-sized electropores on cell membranes.Recently,micro/nano technology has been applied to many novel micro EP devices which can not only significantly increase uptake of biomolecules,DNA transfection and cell viability,but also enable large-scale single-cell EP.However,most EP theories developed in the past three decades can not precisely predict the experimental results of EP of biological cells.With the advanced micro EP chips for large-scale single-cell EP experiments,more precise EP theoretical models can be developed to describe the complicated multiscale dynamic behavior of EP.

Interaction between gene p53 and oncogene mdm2 in human glandularlung cancer cellline GLC82

Objective To study the interaction between oncogene mdm2 and wp53 in human glandular lung cancer cell line GLC 82.MethodsBy lipofectamine mediated DNA transfec^tion, wp53 and mdm2 were transfected separately or co transfected into GLC 82 cells via retrovival vector pDOR neo, a carrier of wp53 and mdm2.Results The growth of GLC 82 cells was blocked and their DNA synthesis inhibited by wp53, its colony forming rate in soft agar culture and the tumorigenicity in nude mice declined and mdm2 antagonized the function of wp53.Conclusion After the recombinant vector pDOR mdm2 was transfected into GLC 82 cells containing wp53, mdm2 partially deprives wp53 of its function of inhibiting the growth of GLC 82 cells.