Methods of enhancing the recovery of plasmid genes from neutralised cell lysate

In this study we have investigated the use of flotation and filtration, singly and combined, to enhance the separation of plasmid containing liquors from neutralised lysates with very different levels of solids. Filtration of crude neutralised lysates, containing roughly 100 g l-1 solids, through various diatomaceous earth and cellulose precoat materials was invariably accompanied by severe loss of plasmid through adsorption and/or absorption. The use of more refined and inert filter aids did not alleviate these problems. The finest filter aid, Celatom FP-1SL, gave the best compromise of filtrate clarity (solids content of 0.05 g l-1) and plasmid purity (71%) and was selected for further studies involving combined use of flotation and filtration. Removing the vast bulk of solids prior to filtration by flotation of the floc and draining of the plasmid liquor beneath, impacted dramatically on the filtration performance. Though systematic reductions in the solids challenge per unit filter area were accompanied by increased flux, elevated levels of solids extrusion, chromosomal DNA and protein contamination were also observed, and losses of plasmid to filter aids were still high. We have observed that increasing the scale of operation during lysis and neutralisation from 0.3 or 0.6 l to 15 l is accompanied by significant improvements in separation of cell debris solids from the plasmid and increased recoveries of the plasmid containing liquor. At the latter scale, the drained liquor contained 80% of the plasmid and the solids content was only 0.2 g l-1

Methods of enhancing the recovery of plasmid genes from neutralised cell lysate

In this study we have investigated the use of flotation and filtration, singly and combined, to enhance the separation of plasmid containing liquors from neutralised lysates with very different levels of solids. Filtration of crude neutralised lysates, containing roughly 100 g l-1 solids, through various diatomaceous earth and cellulose precoat materials was invariably accompanied by severe loss of plasmid through adsorption and/or absorption. The use of more refined and inert filter aids did not alleviate these problems. The finest filter aid, Celatom FP-1SL, gave the best compromise of filtrate clarity (solids content of 0.05 g l-1) and plasmid purity (71%) and was selected for further studies involving combined use of flotation and filtration. Removing the vast bulk of solids prior to filtration by flotation of the floc and draining of the plasmid liquor beneath, impacted dramatically on the filtration performance. Though systematic reductions in the solids challenge per unit filter area were accompanied by increased flux, elevated levels of solids extrusion, chromosomal DNA and protein contamination were also observed, and losses of plasmid to filter aids were still high. We have observed that increasing the scale of operation during lysis and neutralisation from 0.3 or 0.6 l to 15 l is accompanied by significant improvements in separation of cell debris solids from the plasmid and increased recoveries of the plasmid containing liquor. At the latter scale, the drained liquor contained 80% of the plasmid and the solids content was only 0.2 g l-1

Disciplines, Outcomes and Purpose in Social Science Education

The gap between school knowledge and academic knowledge has long been acknowledged. The division of the curriculum into separate academic subjects has sometimes been blamed for this problem. On this reading it would make sense to re-model the curriculum so that teaching is multidisciplinary. However, efforts to achieve this ideal have a poor record in England. In this paper we suggest that the answer to the problem is more likely to lie in the way that the curriculum is built from a careful analysis of the actual outcomes of learning. We briefly outline three projects that are based on this supposition, describing the theoretical underpinnings and the main features of the projects

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone