Efficient mispriming during apolipoprotein E genotyping

A determinação do genótipo da apolipoproteína E (ApoE) é um procedimento clinicolaboratorial comum. O método mais freqüentemente utilizado envolve a amplificação por reação em cadeia de polimerase (PCR) de uma região alvo, seguida pela digestão com enzima de restrição do fragmento resultante, obtendo-se, assim, um modelo de fragmentos de restrição genótipo-específico. Nós descrevemos uma dificuldade inesperada que encontramos durante nossa rotina laboratorial.

Cultivation of Bacillus thuringiensis subsp. Kurstaki S93 in a fed-Batch process: effect of the ratio glucose to yeast extract concentrations in the feeding culture media

The feeding media was studied in a fed-batch process for production of the complex spore-d-endotoxin by B. thuringiensis S93. The microorganism was first cultvated in a initial batch followed by an exponential feeding (m=0,25h(-1)) with concentrated culture media (160 g glucose/L) containing different ratios of glucose and autolysed yeast: 8/7, 8/4 and 8/2 (g/g). The batch culture medium was composed of glucose (8 g/L), autolysed yeast (7g/L) and mineral salts. Sporulation and d-endotoxin production were observed only after the end of feeding. To compare the experiments, batch cultivations were also performed with an initial concentration of 8 g/L of glucose and the same ratio of glucose and autolized yeast. Batch cultivations reached lower concentrations of total biomass and spores than the fed-batch ones and percentagens of sporulation higher than 80 per cente. The 8/7 ratio fed-batch cultivations reached the highest biomass concentration, producing however a very low level of sporulation (27per cent) and virtually no d-endotoxin. Cultivations with 8/4 and 8/2 ratios reached the highest concentrations of spores. In those assays, the maximum spores concentration and the maximum sporulation percentage were 8,3x10(9) spores/mL and 90per cent for the 8/4 ratio and 5,6x10(9) spores/mL and 89per cent for the 8/2 ratio.