限制性内切酶KpnⅠ和EcoRⅠ双酶切条件的优化

The optimization of co-digestion conditions by KpnⅠ /EcoRⅠ

目的探讨KpnⅠ和EcoRⅠ双酶切的优化条件。方法 KpnⅠ和EcoRⅠ不同的酶切体系,酶切含有上述两种限制性内切酶酶切位点的载体pEGFP-Ano2,DNA琼脂糖凝胶电泳检测酶切结果,以获取KpnⅠ和EcoRⅠ双酶切的优化条件。结果 50μL酶切体系,KpnⅠ和EcoRⅠ双酶切的适宜条件:首先在1×L缓冲液情况下,应用1μL KpnⅠ酶切1μg质粒1 h,再加入1×H缓冲液和0.01%BSA,应用1μL EcoRⅠ继续酶切1 h。结论获取KpnⅠ和EcoRⅠ双酶切质粒的适宜条件,为今后应用KpnⅠ和EcoRⅠ双酶切实验提供科学的参考依据。

Objective To investigate the optimum conditions of co-digestion by KpnⅠ/EcoR I.Methods Different digestive systems were selected to digest pEGFP-Ano2,containing the KpnⅠ/EcoR I,then DNA agarose gel electrophoresis was used and enzyme digestion results were observed to get the optimum conditions .Results The optimum conditions of codigestion by KpnⅠ/EcoR I in the 50μL systems appears below：first,in the presence of the 1×Buff-er L,1 μL Kpn I digesting 1μg plasmid for 1 h,then 1μL EcoR I,0.01%BSA and 5μL 10 ×Buffer H were added to enzyme digestion for 1 h.Conclusion The optimum conditions of co-digestion by KpnⅠ/EcoR I were got in our systems,which provided the basis for the co-digestion by KpnⅠ/EcoR I in the future.