Molecular Biology Problem Solver 26. 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. | the amount of overdigestion. Consult the manufacturer s stability information. If the reaction produces extra fragments possibly caused by star activity reduce the reaction time or the amount of enzyme. If the reaction is incomplete individually test each enzyme to determine it s ability to linearize the plasmid. A lack of cutting may indicate an inactive enzyme absence of the expected site or inhibitors in the template preparation. Test the enzyme on a second target as a control. If both enzymes are active and the restriction sites are within several bases of each other there may be a problem cutting close to the end of the fragment. Sequential Enzyme sets that are not compatible for double digests require sequential digestion. Always perform the first digest with the enzyme requiring the lower salt buffer. Either salt or the corresponding 10x reaction buffer may then be added to the reaction and the second enzyme can be used directly. To prevent the first enzyme from exhibiting star activity in the second buffer it is wise to heat inactivate prior to addition of the second enzyme. Addition of BSA reducing agents or detergents has no adverse effects on restriction enzymes and may be safely added as required to the reaction. If the pH requirements between the two enzymes differ by more than pH units or the difference in salt requirement is critical NaCl vs. KCl alcohol precipitation between enzyme treatments is commonly performed. Alternatively drop dialysis see procedure D at the end of this chapter is an option. A strategy that can often save a dialysis step would be to perform the first reaction in a 20 ml volume and then add 80 ml containing 10 ml of the higher salt buffer and enzyme to the initial reaction. The second reaction approximates the standard conditions for that enzyme. Expensive enzymes should be optimized and used first in sequential reactions. When planning to use enzymes from different suppliers first consider their optimal activity by .
