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Molecular Biology Problem Solver 19

Molecular Biology Problem Solver 19. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | 4. Load the appropriate amount of sample. Nothing will impair the quality and yield of a purification strategy more than overloading the system. Too much sample can cause an increase in the viscosity of the DNA preparation and lead to shearing of genomic DNA. If you do not know the exact amount of starting material use 60 to 70 of your estimate. How Can You Maximize the Storage Life of Purified DNA The integrity of purified DNA in solution could be compromised by nuclease pH below 6.0 and above 9.0 heavy metals UV light and oxidation by free radicals. EDTA is often added to chelate divalent cations required for nuclease activity and to prevent heavy metal oxidative damage. Tris-based buffers will provide a safe pH of 7 to 8 and will not generate free radicals as can occur with PBS Miller Thomas and Frazier 1991 Muller and Janz 1993 . Free-radical oxidation seems to be a key player in breakdown and ethanol is the best means to control this process Evans et al. 2000 . Low temperatures are also important for long-term stability. Storage at 4 C is only recommended for short periods days Krajden et al. 1999 . Even though some studies have shown that storage under ethanol is safe even at elevated temperatures Sharova 1977 better stability is obtained at -80 C. Storage at -20 C can lead to degradation but this breakdown is prevented by the addition of carrier DNA. RNA stored in serum has also been shown to degrade at -20 C Halfon et al. 1996 . Another approach for intermediate storage is freeze drying DNA-containing samples intact Takahashi et al. 1995 .The DNA within freeze-dried tissue was stable for 6 months but RNA began degrading after 10 weeks of storage. The control of moisture and temperature had a significant effect on shelf life of samples. The long term stability of DNA-containing samples is still being investigated Visvikis Schlenck and Maurice 1998 but some companies offer specialized solutions e.g. RNA Later from Ambion Inc. allowing storage at room .