Encyclopedia of Experiments: Biology
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Fijación de etanol y tinción dapi: un método para visualizar el ADN en C. elegans
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- Comience con una gota de solución de amortiguación en una diapositiva del microscopio y transfiera gusanos intactos a la gota. Una vez que los gusanos estén en su lugar, utilice un tejido libre de pelusas para absorber y eliminar el búfer.
Una vez que el etanol se haya evaporado después del enjuague final, agregue una solución que contenga el tinte ácido nucleico, DAPI, a la muestra. DAPI se une preferentemente a las bases de adenina y timina en la ranura menor del ADN.
A continuación, agregue un conservante anti-fade para prolongar la vida útil de la muestra. Aplicar un resbalón de cubierta y sellarlo a la diapositiva. A continuación, utilizar un microscopio fluorescente con un filtro azul para visualizar los cuerpos DAPI, que, dependiendo de la célula y su estado de ciclo, puede representar un solo cromosomas, pares de cromatados hermanos, o núcleos enteros. En este experimento, contaremos pares de cromosótidos hermanos manchados dapi en omacitos, para identificar cepas tetraploides de Caenorhabditis elegans.
- Las cepas tetraploides tienen 12 pares cromosómicos, que se pueden validar contando los cuerpos manchados dapi en ocitos no fertilizados. Para ello, deje caer de cinco a diez microlitros de amortiguador M9 en un tobogán y transfiera de seis a 10 tetraploides putativos en la gota.
A continuación, baje 10 microlitros de 90% de etanol sobre los gusanos y observe cómo el etanol se evapora por completo. Tan pronto como termine la evaporación, repita el proceso de agregar etanol y verlo evaporarse.
Para el almacenamiento a largo plazo de las diapositivas, monte los gusanos en un anti-fundido comercialmente disponible o casero.
En primer lugar, encontrar el ócito no fertilizado más maduro, que es inmediatamente adyacente a la espermatética, y aún no ha entrado ni en la espermatética ni en el útero. Los cuerpos DAPI aquí son presumiblemente pares cromosómicos individuales.
Los ocitos de tipo salvaje tienen seis cuerpos DAPI en promedio. La presencia de 12 cuerpos DAPI indica que los animales de esta cepa son tetraploides parciales o completos.
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