By Leonard Davis (Auth.)
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Additional resources for Basic Methods in Molecular Biology
P u t e a c h agarose (typically 20 x 20 cm) gel in a plastic b o x with 500 ml of solution A at r o o m t e m p e r a t u r e . Agitate periodically by hand. After 30 min, replace with 500 ml of fresh solution A. Continue periodic shaking for a n o t h e r 30 min. This s t e p is designed to d e n a t u r e double-stranded DNA. 2 . To r e t u r n gel t o a m o r e neutral pH, replace liquid with 500 ml of solution B. S h a k e periodically, a s above, for 30 min. Replace with 500 ml of fresh solution Β a n d s h a k e periodically for 30 min.
T w o types of p r o b e s can b e synthesized: 1. If t h e sequence of t h e DNA or RNA of interest is known, an e x a c t comple m e n t a r y p r o b e can b e synthesized. 2 . If t h e starting point for determining the sequence of t h e p r o b e is from t h e amino acid sequence of a protein product, it is not possible t o synthesize a single p r o b e of significant length with complementary sequence to t h e RNA that uniquely c o d e s for t h a t protein, due to t h e degeneracy of t h e genetic code.
5 Diagram of Xgtll, showing major RE sites. SECTION EMBL3 and EMBL4 EMBL3 is a λ r e p l a c e m e n t vector carrying t w o polylinker sequences that facili tate t h e cloning of Mfeol, BamHI, EcoRl, Bglll, Xhol Bell, and Sail cut fragments. It is m o s t useful a s a cloning vector for generating genomic libraries after partial Mbol digestion. This cloning vector can a c c o m o d a t e u p to approximately 23-kb inserts, making it suitable for genomic library construction. The design of t h e polylinker s e q u e n c e s simplifies preparation of t h e vector for cloning partial Mbol fragments 15-20 kb in size.
Basic Methods in Molecular Biology by Leonard Davis (Auth.)