GMP plasmid DNA can be used as starting materials by companies which manufacture ATMPs (advanced therapy medicinal products) such as viral vectors (e.g. lentivirus and AAV) or in vitro transcribed-RNA (IVT-RNA) (e.g. mRNA, long RNA) Plasmid DNA can also be the active pharmaceutical ingredi...
Extraction of plasmid DNA by alkaline lysis(碱裂解法抽提质 粒DNA) Experiment 1. Extraction of plasmid DNA by alkaline lysis [experimental principle] Plasmids are small, double stranded, ring-shaped DNA found in vitro, ranging in size from 1-200kb to autonomously replicating in host bacteria. In...
restriction analysis and sequence data. Third, the basis for nearly all modern methods of plasmidDNA extractionwas established by HC Birnboim and J Doly[5]withalkaline lysisofbacterial cellsfollowed by neutralization with potassium acetate precipitating the chromosomal DNA in a complex with other cell...
still numerous human clinical trials are performed. Clinical progress in the field of DNA vaccination and gene therapy has led to an increased demand of pharmaceutical-grade plasmid DNA (pDNA), manufactured under the conditions of current Good Manufacturing Practices (cGMP). Consequently...
Apparatus and methods are described for pharmaceutical grade manufacture extrachromosomal nucleic acids from cell lysates using flotation to separate and eliminate undesired insoluble cellular debris including chromosomal DNA from the lysates. A gas is introduced to controllably generate bubbles that reduce ...
The hepatocytes located in bottom after centrifuge at 200g for 10min were collected for genomic DNA extraction. Lung transvascular albumin flux assessment The EBA flux assay was carried out as described previously (Huang et al., 2016). Briefly, Evans blue dye (Sigma Aldrich) was dissolved in ...
production processes plasmid DNA is also interesting as an alternative to viral delivery systems because the manufacture, storage, and application of this product poses fewer process and quality control problems (Schleef, 2001). However, the problems regarding in- ...
Site-directed and saturation mutagenesis are critical DNA methodologies for studying protein structure and function. For plasmid-based gene mutation, PCR and overlap-extension PCR involve tedious cloning steps. When the plasmid size is large, PCR yield may be too low for cloning; and for saturation...
plasmid DNA into the three different forms, supercoiled (form I), nicked circle (form II), and linearized (form III), and the desired plasrnid form is collected. Further extraction with butanol is required to remove residual ethidium bromide followed by DNA precipitation using alcohol. Additional...
amplification of DNA fragments for cloning was performed using the high-fidelity Pfu Turbo™ polymerase (Stratagene™). Other PCR amplification was performed using Taq DNA polymerase (Life Technologies™). DNA fragments were isolated from agarose gels using the QIAquick gel extraction kit from ...