Abstract
A eficácia dos protocolos de extracção de ADN podem ser altamente dependente tanto do tipo de amostra a ser investigada e os tipos de análises realizadas a jusante. Considerando-se que a utilização de novas técnicas de análise da comunidade bacteriana (por exemplo, microbiomics, metagenómica) está se tornando mais prevalente nas ciências agrícolas e ambientais e muitas amostras ambientais dentro dessas disciplinas pode ser physiochemically e microbiologicamente único (por exemplo, fezes e amostras de maca / cama de o espectro de produção de aves), métodos de extração de DNA apropriadas e efetivas precisam ser cuidadosamente escolhido. Portanto, um novo método semi-automatizado de extração de DNA híbrido foi desenvolvido especificamente para uso com amostras de produção de aves ambiental. Este método é uma combinação dos dois tipos principais de extracção de ADN: mecânica e enzimática. A intensa etapa de homogeneização mecânica de duas etapas (usando talão batendo formulado especificamente para ambientesAmostras Tal) foi adicionado ao início do método "padrão de ouro" de extracção de ADN enzimático para as amostras fecais para melhorar a remoção de bactérias e ADN a partir da matriz da amostra e melhorar a recuperação dos membros Gram-positivas comunitárias bacterianas. Uma vez que a porção de extração enzimática do método híbrido foi iniciada, o restante processo de purificação foi automatizado usando uma estação de trabalho robótica para aumentar a produtividade de amostras e diminuir o erro de processamento da amostra. Em comparação com os rigorosos métodos de extração de DNA mecânicos e enzimáticos, este método híbrido novela proporcionou o melhor desempenho global combinada ao considerar quantitativa (usando 16rRNA qPCR) e qualitativa (usando microbiomics) estimativas do total de comunidades bacterianas ao processar fezes de aves e amostras de serapilheira .
Materials
| Name | Company | Catalog Number | Comments |
| Lysing Matrix E tube | MPBio | 6914-050 | Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1,000 tubes) |
| Sodium Phosphate Solution | MPBio | 6570-205 | Can be purchased individually, or also contained within the FastDNA Spin Kit for feces (Cat. No. 116570200) |
| PLS Buffer | MPBio | 6570-201 | |
| Buffer ASL (560 ml) | Qiagen | 19082 | |
| FastPrep 24 homogenizer | MPBio | 116004500 | 48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step |
| QIAamp DNA Stool Mini Kit | Qiagen | 51504 | |
| QIAcube24 (110V) | Qiagen | 9001292 | Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done. |
| Filter-Tips, 1,000 ml (1024) | Qiagen | 990352 | |
| Filter-Tips, 200 ml (1024) | Qiagen | 990332 | |
| QIAcube Rotor Adapters (10 x 24) | Qiagen | 990394 | For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative. It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690 |
| Sample Tubes RB (2 ml) | Qiagen | 990381 | Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352 |
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