Browsing by Subject "Purification"

Now showing 1 - 4 of 4
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    Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Silva Almeida Vicente, Anna Luiza; Alves Bianchini, Raquel; Laus, Ana Carolina; Macedo, Graziela; Reis, Rui Manuel; Vazquez, Vinicius de Lima
    Melanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffinembedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.
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    Development of fast and simple methods for porcine haptoglobin and ceruloplasmin purification
    (Murcia, Universidad de Murcia, Servicio de Publicaciones, 2010) Fuentes, P.; Gutiérrez Andrés, M.; Soler, L.; Cerón Madrigal, José Joaquín; Martínez Subiela, Silvia; Facultad de Veterinaria
    Two fast and simple methods for porcine haptoglobin (Hp) and ceruloplasmin (Cp) purification are described in this paper. Hp was purified by ammonium sulphate fractionation and a FPLC Superdex-200 gel chromatography. The protein obtained showed two bands with a Mr (molecular mass) of about 44 kDa and 12.9 kDa corresponding to heavy (ß) and light (a) chains of Hp respectively, on sodium dodecyl sulphate-polyacrilamide gel electrophoresis, under reducing conditions. Ceruloplasmin (Cp) was isolated by one step of chromatography on amino-ethyl-derivatized Sepharose followed by gel filtration on a Superdex-200 column. The Mr of the protein, as estimated by SDS-PAGE was approximately 150 kDa. In conclusion, two new protocols have been developed for porcine Hp and Cp purification, being less time-consuming and technically demanding than those previously reported. This paper could represent an interesting guideline and be of help to obtain pure protein to use as specie-specific standard material and to produce specific antibodies.
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    Open Access
    Recycled Sewage: A Water Resource for Dry Regions of Southeastern Spain
    (Springer Nature, 2018-11) Gil Meseguer, Encarnación; Bernabé Crespo, Miguel Borja; Gómez Espín, José María; Geografía
    The latitude and layout of the Betic orography make southeastern Spain one of the driest climatic regions in Europe. Most of its territory is part of the Segura Hydrographic Demarcation (DHS). The DHS features a water resources vs water demands deficit equal to 480 hm3/ year (1 hm3 = 100 m3) during the 2009–2015 hydrologic planning period. A new paradigm for water policy in Spain has emerged for the hydrological planning period (2016–2021), which calls for a greater contribution of unconventional resources (desalination and reuse of municipal sewage). The investment made in the DHS, in terms of sewage purification and regeneration, produces about 110 hm3/year of purified sewage annually. Irrigation is the main consumer of these reuse flows. Irrigation districts develop conveyance and storage infrastructure to import treated sewage from Sewage Treatment Plants (STPs) and Water Regeneration Stations (WRSs). Anthropogenically-caused climate change has brought additional stress on surface water and groundwater, thus making water recycling an important component of the water supply portofolio in Spanish arid regions. Recycled water increases the resources of semi-arid regions (up to 10% of total resources), like in the Southeast of Spain. It is of great social value as it contributes to water safety, economic dynamism and biodiversity. Investment made in this sector and public policies make possible the implementation of recycling system, turning this limited resource into a social, political and economic interest, reaching levels of 99.5% purified and 97% reused in Murcia. Similar regions could import this management system and the concessionary model of reclaimed water. In dry regions, these water management models make recycled water, rather than an alternative, a significant complement to local water resources
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    Significado y función de los baños en la novela griega.
    (Universidad de Murcia, Servicio de Publicaciones., 2025) Martín Ventas, Paula; Sin departamento asociado
    Tras explicar la simbología del agua y del baño en la Antigua Grecia a través de algunas leyendas y ritos en los que la inmersión en el agua se emplea, entre otras cosas, como método de lustración o como medio para llevar a cabo la transformación de ciertos individuos, esta investigación se centrará en las funciones que cumplen los baños en las novelas de Caritón de Afrodisias, Longo, Aquiles Tacio y Heliodoro: ser un método de purificación previo a prácticas religiosas o posterior al contacto con los muertos, demostrar la inocencia de una persona, servir de elemento erótico, señalar puntos importantes de la trama y ser uno de los constituyentes de ciertos ritos de tránsito.