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By Leonard Davis (Auth.)

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7 Xhol BomHl Hindis EcoRl Bg/U Sma I BomHl Hindis. 4 3 . 4 Diagram of XgtlO, showing major RE sites. 7-kb linear double-stranded X bacteriophage cloning vector de­ signed exclusively for cloning small EcoRl fragments (less t h a n 6 kb), in particu­ lar cDNA fragments with EcoRl linkers. The unique EcoRl site u s e d for inserting foreign DNA is located n e a r t h e COOH t e r m i n u s of t h e lac Ζ gene. Foreign DNA s e q u e n c e s in this cloning vehicle can b e e x p r e s s e d as β-galactosidase fusion proteins.

1 2 . Centrifuge for 10 min at 12,000 x g to collect precipitated DNA. Wash pellet in 5 ml of 80% ethanol. Collect t h e pellet by centrifugation. D e c a n t t h e ethanol and air-dry t h e pellet briefly. 1 3 . 5 ml of TE buffer. Allow pellet t o dissolve overnight at 37°C with shaking, if DNA is difficult to r e s u s p e n d . 2 All tubes must be extremely clean. , Corex) with 50% nitric acid before using to remove any DNA from tubes. Rinse thoroughly and autoclave before use. 46 5 I Preparation of DNA from Eukaryotic Cells Next Day 14.

Flip gel over, a n d r e m o v e t h e t o p glass p l a t e , a n d slide gel o n t o wick. Align gel w i t h e d g e of wick a n d p l a t e . S m o o t h gel a n d r e m o v e t r a p p e d bubbles b y rolling a glass p i p e t t e o v e r surface (see Figure 5-3B). N o t e : Gel is n o w u p s i d e - d o w n , b e c a u s e it is b e s t t o blot from t h e b o t t o m for s h a r p e r resolution a n d t o maintain t h e left-to-right sample orientation o n t h e b l o t t e d NC filters.

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