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        标准连接反应

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        1851
         
        1. Prepare a ligation mix:

          Ligation Mix (2x)标准连接反应   10x ligase buffer1.0 mldigested vector
          (0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 ml标准连接反应
           
        2. divide ligation mix into two Eppendorf tubes

          Ligation Rxn 标准连接反应  InsertControlligation mix4.0 ml4.0 mlinsert1.0 ml--- mlT4 DNA ligase (400 u/ml)0.2 ml0.2 mlTotal5.2 ml4.2 ml标准连接反应
           
        3. incubate for 2-3 h (or ovn) at 14°C
        4. proceed with the transformation of the appropriate E. coli strain

         

        Solutions:
         

        10x Ligation Buffer:

        0.5 M Tris-HCl pH 7.8, 50 mM MgCl2, 0.1 M b-mercaptoethanol, 50 mM ATP, 5 mg/ml BSA

        Ligation Buffer (10x)标准连接反应   1 M Tris-HCl pH 7.8500.0 ml1 M MgCl250.0 mlb-mercaptoethanol7.0 ml100 mM ATP50.0 ml0.1 g/ml BSA50.0 mlH2O343.0 mlTotal1 ml标准连接反应     
        Remarks:

        As usually when pipetting enzyme reactions keep the tubes on ice during all manipulations. Cip-treat a digested vector cut with only a single enzyme. If the vector was double-digested: phenolize prior to ligating. Use equimolar amounts of vector and insert. As a rule we use 50 ng vector (3 kb) and 50 ng insert (3 kb). More values are given in the table below. Recalculate accordingly when using different-length vectors. Don't use less than 8 ng or so for fragments smaller than 0.5 kb. In the case of small fragments circularization occurs and removes the F from the rxn.
         

        F Used in Standard Ligation标准连接反应 vector (50 ng, 3kb)Length of F
        (kb)Amount of F (ng)0.58.3116.71.525233.32.541.73503.558466.7583.361007116.3标准连接反应

         

        Materials:Reagent/ToolSupplierCat.-#标准连接反应 BSA  ATP  T4 DNA Ligase  标准连接反应
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