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Joseph Sambrook – One of the best experts on this subject based on the ideXlab platform.
Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps.Cold Spring Harbor protocols, 2018Co-Authors: Michael R Green, Joseph SambrookAbstract:
In recent years, with the advent of polymerase chain reaction and the development of highly efficient methods of cloning and DNA sequencing, the need to prepare large quantities of plasmid vectors and recombinants has greatly diminished. In consequence, this protocol, at one time in common use, has been largely replaced by faster and easier column-based purification methods. In this protocol, plasmid DNA is purified from the cleared bacterial lysate by centrifugation to equilibrium in CsCl gradients containing ethidium bromide. These gradients have an aesthetic quality that justifies the continued presentation of a protocol that in many other respects is an antique. The typical yield of high-copy-number plasmid vectors or of amplified low-copy-number vectors prepared by this method is ∼3-5 µg of DNA/mL of original bacterial culture. The yield of recombinant plasmids containing inserts of foreign DNA is usually slightly lower, depending on the size and nature of the cloned DNA fragment.
Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreps.Cold Spring Harbor protocols, 2016Co-Authors: Michael R Green, Joseph SambrookAbstract:
In this protocol, plasmid DNA is isolated from small-scale (1-2 mL) bacterial cultures. Yields vary between 100 and 5 µg of DNA, depending on the copy number of the plasmid. Miniprep DNA is sufficiently pure for use as a substrate or template in many in vitro enzymatic reactions. However, further purification is required if the plasmid DNA is used as the substrate in sequencing reactions.
Jae Hyeon Park – One of the best experts on this subject based on the ideXlab platform.
extraction of sporopollenin exine capsules from sunflower pollen grainsRSC Advances, 2016Co-Authors: Raghavendra C Mundargi, Michael G Potroz, Jae Hyeon ParkAbstract:
Sporopollenin exine capsules (SECs) are highly robust natural microscale capsules that can be extracted from plant spores and pollen grains, albeit through complex processing schemes. Herein, we report new insights into pollen processing by Alkaline Lysis and acidoLysis with various process conditions. Alkaline Lysis of sunflower pollen grains damages the unique pollen microstructure and acidoLysis enables us to devise a simple process to extract SECs from sunflower pollen grains with a uniform particle size distribution. The SECs retain the natural morphology, offering an improved general scheme to streamline pollen processing for biomaterial applications.
eco friendly streamlined process for sporopollenin exine capsule extractionScientific Reports, 2016Co-Authors: Raghavendra C Mundargi, Michael G Potroz, Jae Hyeon ParkAbstract:
Sporopollenin exine capsules (SECs) extracted from Lycopodium clavatum spores are an attractive biomaterial possessing a highly robust structure suitable for microencapsulation strategies. Despite several decades of research into SEC extraction methods, the protocols commonly used for L. clavatum still entail processing with both Alkaline and acidoLysis steps at temperatures up to 180 °C and lasting up to 7 days. Herein, we demonstrate a significantly streamlined processing regimen, which indicates that much lower temperatures and processing durations can be used without Alkaline Lysis. By employing CHN elemental anaLysis, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and dynamic image particle anaLysis (DIPA), the optimum conditions for L. clavatum SEC processing were determined to include 30 hours acidoLysis at 70 °C without Alkaline Lysis. Extending these findings to proof-of-concept encapsulation studies, we further demonstrate that our SECs are able to achieve a loading of 0.170 ± 0.01 g BSA per 1 g SECs by vacuum-assisted loading. Taken together, our streamlined processing method and corresponding characterization of SECs provides important insights for the development of applications including drug delivery, cosmetics, personal care products, and foods.
Peter Dunnill – One of the best experts on this subject based on the ideXlab platform.
Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease AJournal of biotechnology, 2001Co-Authors: G.d. Cooke, Peter Dunnill, R.m. Cranenburgh, J.a.j. Hanak, D.r. Thatcher, John M. WardAbstract:
Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell Lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMR-NaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by Alkaline Lysis. The RNaseA protein is stable throughout incubation at high pH (similar to 12-12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following Alkaline Lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18 Delta lacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements. (C) 2001 Elsevier Science B.V. All rights reserved.
Rheological properties of chromosomal and plasmid DNA during Alkaline Lysis reactionBioprocess Engineering, 1999Co-Authors: L A S Ciccolini, Nigel J. Titchener-hooker, Parviz Ayazi Shamlou, John M. Ward, Peter DunnillAbstract:
The recovery of plasmid DNA from cells is achieved by a two-step chemical reaction which usually employs sodium hydroxide and sodium dodecyl sulphate followed by neutralisation with a chilled solution of potassium acetate. It is important that the gelatinous floc of chromosomal DNA with proteins debris is not broken down since fragments of chromosomal DNA are difficult to separate from plasmid DNA. To accomplish the operation at scale demands knowledge of the rheology as the reactions proceed. In this paper a co-axial cylinder rheometer is used to record the changes for two strains of Escherichia coli cells. AnaLysis of the liquor, prior to neutralisation indicates that at shear rates below 367 s−1 the flow properties during Lysis are non-Newtonian, but above 367 s−1 flow behaviour becomes Newtonian. Additionally, following neutralisation, the rheological data show that the clear liquor obtained at low shear levels is Newtonian, but the gelatinous floc has strong viscoelasticity. These properties strongly influence subsequent stages for the removal of solids.
Time Course of SDS–Alkaline Lysis of recombinant bacterial cells for plasmid releaseBiotechnology and bioengineering, 1998Co-Authors: Laura A. S. Ciccolini, Nigel J. Titchener-hooker, Parviz Ayazi Shamlou, John M. Ward, Peter DunnillAbstract:
SDS-Alkaline Lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of Lysis reaction. The results of the experiments showed that cell Lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.