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1、takara宝生物公司限制酶双酶切buffer表Enzyme Acc I BamH EcoR EcoR Hinc Bgl II Cla I I I V II 10 M 10 H 10 H 10 M Hind Kpn I Nco I Nde I Not I III 10 10 10 10 L K M H +BSA Pst I Pvu I Sac I Sal I Sma I Supplied 10 10 10 K Buffer M H Acc I BamH I Bgl II Cla I EcoR I EcoR V Hinc II Hind III Kpn I - 0.5 K 10 H 10 10
2、10 10 T +BSA K 10 L H H +BSA +Triton +BSA 0.5 0.5 1 M 0.5 K 0.5 1.5 1 T 1 T 1 M 1 M 1 M 1 M 1 M 1 T 1 M 1 M K K +BSA +BSA K T +BSA - 1 K 1 K 1 K 1 K - 0.5 0.5 1 K 0.5 K 0.5 1.5 1 K 1 K 1 K 1 K K K +BSA +BSA K T 0.5 T +BSA 1 T 1 K 1 H 1 H 1 H 2 K 1 K 1 T - 1 H 1 H 1 M 1 M 1 M - 1 H 1 M 1 M 1 M - 1 K
3、1 H 0.5 1 T 1 H 1 H 1 K 1 H +BSA +BSA K +BSA 1 K 1 H 1 T 1 H 1 H 1 K 1 M 1 H +BSA +BSA +BSA 1 K 1 H 1 T 1 H 1 H 1 K 1 M 1 H +BSA +BSA +BSA 0.5 K +BSA 1 M 1 K 1 H 1 M 1 K 1 H 1 H 0.5 1 K 1 H 1 H 1 H K 1 M 0.5 1 K 1 H 0.5 2 T 1 K 1 H 1 H 1 K 1 H K +BSA +BSA K - 1 M 1 M - 1 M - 0.5 K +BSA 0.5 2 K 1 M 1
4、 M 2 T K 1 M 0.5 K 0.5 1.5 1 T 1 T 1 M 1 M +BSA +BSA K K +BSA 1 K 0.5 K 1.5 1 T 1 K 1 M 1 K 1 M +BSA +BSA K +BSA 0.5 1.5 0.5 K 0.5 1 T K 1 T 1 M 1 L T +BSA K +BSA +BSA +BSA - 1 K +BSA 1 K 0.5 K 1 K 1 K +BSA +BSA +BSA +BSA - 0.5 1.5 1 T K T +BSA +BSA +BSA 1 M 1 K 1 K 1 M 1 M 1 K 1 M 0.5 0.5 1 M 1 T 1
5、 M 1 M 1 M 1 M K K 1 M 1 K 1 K 1 K 1 K 1 K 1 M 1 K +BSA +BSA +BSA +BSA +BSA +BSA +BSA +BSA Nco I Nde I Not I 1 T 1 K 1 H 1 H 1 H 1 H 1 T 1 K 1 T 1 H 1 T 1 H 1 K 1 T 1 H +BSA +BSA - 1 H 2 K +BSA +BSA - 0.5 0.5 1 H K T +BSA +BSA +BSA 0.5 0.5 0.5 0.5 0.5 0.5 1 H 1 H 1 H 1 H 1 H K K K K K K +BSA +BSA +B
6、SA +BSA +BSA +BSA +BSA +BSA +BSA +BSA +BSA Pst I 1 K 1 H 1 M 1 K 1 H 1 H 1 H 1 H 1 M 1 M 1 M 1 H +BSA +BSA 0.5 1 K 1 M 1 H T +BSA - 0.5 K - 1.5 1 K K +BSA +BSA 1.5 1 T T +BSA +BSA - 1.5 T +BSA - Pvu I 0.5 0.5 0.5 1 K 2 K 1 K 1 K 1 K 1 K 1 K 1 K 1 K 1 K K K K +BSA +BSA 0.5 0.5 0.5 1 M 1 M 1 M 1 M 1 M
7、 1 L K K K 1.5 1.5 1.5 1.5 1 H 1 H 1 H 1 H T T K K Sac I 0.5 0.5 K 0.5 K 1 T 1 M +BSA K +BSA Sal I Sma I 1.5 1.5 1.5 1.5 1 H T T 1 H 1 H K T +BSA +BSA +BSA +BSA +BSA 1 T 0.5 1 T 1 T 1 T 0.5 1 T 1 T 1 T 1 T 1 T 0.5 T 0.5 1 K 1 T 1.5 +BSA T +BSA +BSA +BSA K +BSA +BSA +BSA +BSA +BSA +BSA T +BSA +BSA T
8、+BSA Spe I Sph I Xba I Xho I +BSA +BSA +BSA 1 M 1 K 1 H 1 M 1 H 1 H 1 M 1 M 1 M 1 K 1 H 1 T 1 H 1 H 1 K 1 M 1 H +BSA +BSA +BSA 0.5 T +BSA 0.5 0.5 1 K 1 H 0.5 1 K 1 H 1 H 1 H 1 H 2 T 1 K 1 H 1 H 1 K 1 H K K +BSA +BSA K 1 M 0.5 1 M 0.5 K 0.5 1.5 1 T 2 T 1 M 1 M 2 T 1 M 1 M 1 M 1 T 1 M 1 M K +BSA +BSA
9、K T +BSA 1 K 1 H 1 T 1 H 1 H 1 K 1 M 1 H +BSA +BSA +BSA 1 M 1 K 1 H 1 H 1 H 1 H 1 M 1 M 1 M Note: 1) It is confirmed that 10 units of each enzyme completely digest 1 g of DNA at 37C in one hour in 50 l reaction mixture. 2) The concentration of Glycerol should be less than 10% to minimize star activity. 3) DNA may not be digested completely, when recognition sequences of two enzymes are close each other, or when DNA takes high-structure conformation. 反应体系： 10* M Buffer 质粒T-5.3kbGt1 Xba、Hind 无菌水 37水浴保温2h 2ul 3ul 各0.5ul 14ul
