Encyclopedia of Experiments: Biology
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Etanol Fiksasyonu ve DAPI Boyama: C. elegans'ta DNA'yı Görselleştirmek için Bir Yöntem
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- Mikroskop kaydırağına bir damla tampon çözeltisi ile başlayın ve bozulmamış solucanları damlacığına aktarın. Solucanlar yerleştirildikten sonra, tamponu emmek ve çıkarmak için tüy bırakmayan bir doku kullanın. Numuneleri birkaç kez% 90 etanol ile yıkarak, her uygulamadan sonra buharlaşmasını sağlayarak sabitleyin.
Etanol son durulamadan sonra buharlaştıktan sonra, numuneye nükleik asit boyası DAPI'yı içeren bir çözelti ekleyin. DAPI, DNA'nın küçük oluğundaki adenin ve timin bazlarına tercihen bağlanır. Bağlandığında, DAPI-DNA kompleksi UV ışığını emer ve görünür mavi ışık yayar.
Ardından, numunenin raf ömrünü uzatmak için solmaya karşı koruyucu ekleyin. Daha sonra, hücreye ve döngü durumuna bağlı olarak tek kromozomları, kardeş kromatid çiftlerini veya tüm çekirdekleri temsil edebilecek DAPI gövdelerini görselleştirmek için mavi filtreli bir floresan mikroskop kullanın. Bu deneyde, Caenorhabditis eleganlarının tetraploid suşlarını tanımlamak için Oositlerdeki DAPI lekeli kardeş kromat çiftlerini sayacağız.
- Tetraploid suşları, DAPI lekeli gövdeleri kısırlaştırılmamış oositlerde sayarak doğrulanabilen 12 kromozom çiftine sahiptir. Bunu yapmak için, beş ila on mikrolitre M9 tamponunu bir kaydırağa bırakın ve damlaya altı ila 10 putatif tetraploid aktarın.
Daha sonra solucanların üzerine % 90 etanolden 10 mikrolitre bırakın ve etanolün tamamen buharlaşmasını izleyin. Buharlaşma biter bitmez, etanol ekleme ve buharlaşmasını izleme işlemini tekrarlayın. Toplamda, dört uygulamada 10 mikrolitre ekleyin. Son damla buharlaştıktan sonra, mikrolitre başına iki nanogramda altı mikrolitre DAPI veya benzer bir leke ekleyin.
Slaytların uzun süreli depolanabilmesi için solucanları piyasada bulunan veya ev yapımı bir anti-solmaya monte edin. Sonra bir kapak ucu ekleyin ve kenarları oje ile kapatın. Oje kuruduktan sonra, slaytlar puanlanabilir. Floresan mikroskop ve 100 x büyütme kullanın.
İlk olarak, spermatheca'ya hemen bitişik olan en olgun kısırlaştırılmamış oosit bulun, ve henüz spermatheca veya uterusa girmedi. Buradaki DAPI gövdeleri muhtemelen tek kromozom çiftleridir. Sonra, oosit çekirdeğine odaklanın ve DAPI gövdelerini sayarken, yavaşça taramak için mikroskobun ince odağını kullanın.
Yabani tip oositlerin ortalama altı DAPI gövdesi vardır. 12 DAPI gövdesinin varlığı, bu suşun içinde hayvanların kısmi veya tam tetraploidler olduğunu gösterir. Suş başına en az 10 hayvanı analiz edin. Genellikle kromozom çiftleri çok yakın veya dokunaklı olacaktır, bu nedenle DAPI cisimlerinin sayısı genellikle gerçek kromozom çiftlerinin sayısından daha azdır.
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