Encyclopedia of Experiments: Biology
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Fissazione dell'etanolo e colorazione DAPI: un metodo per visualizzare il DNA in C. elegans
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- Iniziare con una goccia di soluzione tampone su uno scivolo al microscopio e trasferire i vermi intatti sulla goccia. Una volta che i vermi sono in posizione, utilizzare un tessuto privo di pelucchi per assorbire e rimuovere il tampone. Fissare il campione bagnando i campioni con il 90% di etanolo più volte, consentendogli di evaporare dopo ogni applicazione.
Una volta che l'etanolo è evaporato dopo il risciacquo finale, aggiungere una soluzione contenente il colorante acido nucleico, DAPI, al campione. DAPI si lega preferenzialmente alle basi di adenina e timina nel solco minore del DNA. Quando è legato, il complesso DAPI-DNA assorbe la luce UV ed emette luce blu visibile.
Aggiungere quindi un conservante anti-dissolvenza per prolungare la durata di conservazione del campione. Applicare uno slittamento di copertura e sigillare la diapositiva. Utilizzare quindi un microscopio fluorescente con un filtro blu per visualizzare i corpi DAPI che, a seconda della cellula e del suo stato di ciclo, possono rappresentare cromosomi singoli, coppie cromatidi sorelle o nuclei interi. In questo esperimento, conteremo coppie cromatidi sorelle macchiate DAPI negli ovociti, per identificare ceppi tetraploidi di elegans caenorhabditis.
- I ceppi tetraploidi hanno 12 coppie cromosomiche, che possono essere convalidate contando i corpi macchiati DAPI in ovociti non fecondati. Per fare ciò, far cadere da cinque a dieci microlitri di tampone M9 su uno scivolo e trasferire da sei a 10 tetraploidi putativi nella goccia. Sotto un microscopio sezionante, immergere con cura la maggior parte dell'M9 in un tessuto di pulizia privo di pelucchi.
Quindi far cadere 10 microlitri di etanolo al 90% sui vermi e guardare l'etanolo evaporare completamente. Non appena termina l'evaporazione, ripetere il processo di aggiunta di etanolo e guardarlo evaporare. In totale, aggiungere 10 microlitri in quattro applicazioni. Dopo che l'ultima goccia evapora, aggiungere sei microlitri di DAPI a due nanogrammi per microlitro o una macchia simile.
Per la conservazione a lungo termine delle diapositive, montare i vermi in un anti-dissolvenza disponibile in commercio o fatto in casa. Aggiungere quindi un coverslip e sigillare i bordi con smalto per unghie. Dopo che lo smalto per unghie si è asciugato, è possibile segnare le diapositive. Utilizzare un microscopio fluorescente e un ingrandimento 100 x.
In primo luogo, trovare l'ovocita non fecondato più maturo, che è immediatamente adiacente alla spermateca, e non è ancora entrato né nella spermateca né nell'utero. I corpi DAPI qui sono presumibilmente singole coppie cromosomiche. Successivamente, concentrati sul nucleo dell'ovocita e usa la messa a fuoco fine del microscopio per scansionarla lentamente, dall'alto verso il basso, contando i corpi DAPI. Quindi riconta i corpi DAPI nello stesso nucleo spostando la messa a fuoco dal basso verso l'alto.
Gli ovociti di tipo selvatico hanno in media sei corpi DAPI. La presenza di 12 corpi DAPI indica che gli animali in questo ceppo sono tetraploidi parziali o completi. Analizzare almeno 10 animali per ceppo. Spesso le coppie cromosomiche saranno molto vicine o toccanti, quindi il numero di corpi DAPI è spesso inferiore al numero effettivo di coppie cromosomiche.
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