Ligation   

     Vector(CIP) and insert (fill-in) are purification by phenol/chloroform  

  condition
     1. reaction solution
        10× buffer             1
        PEG4000                1
        T4 ligase                 1
             total                   3

     3 reactions

  vectors

  inserts

   H2O

  reaction solution

     total

       control

       2

      0

     5

             3

      10

     ligation-1

       2

      2

     3

             3

      10

     ligation-2

       2

      4

     1

             3

      10

     2.incubate the 3 reaction in 22°C   water bath for 2 hours.