标准连接反应

1. Prepare a ligation mix:
   
   Ligation Mix (2x)   10x ligase buffer1.0 mldigested vector
   (0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 ml
    
2. divide ligation mix into two Eppendorf tubes
   
   Ligation Rxn  InsertControlligation mix4.0 ml4.0 mlinsert1.0 ml--- mlT4 DNA ligase (400 u/ml)0.2 ml0.2 mlTotal5.2 ml4.2 ml
    
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)   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     

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 vector (50 ng, 3kb)Length of F
(kb)Amount of F (ng)0.58.3116.71.525233.32.541.73503.558466.7583.361007116.3

 

Materials:Reagent/ToolSupplierCat.-# BSA  ATP  T4 DNA Ligase