Ligation   

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

  condition
     1. reaction solution
        10¡Ñ 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¢XC   water bath for 2 hours.