Ligation of Sticky Ends with T4
Thaw the T4 DNA Ligase Buffer and resuspended at room temperature .
Set up the following reaction mixture at room temp : 10x , T4 DNA Ligase Buffer ; 2 ul , Vector DNA ; 0.020 , pmol .
Insert DNA , 0.060 pmol ; Nuclease - free Water to 20 ul ; T4 DNA Ligase , 1 ul .
Gently mix the reaction by pipetting up and down and microfuge briefly .
For sticky ends , incubate at 22 C for 10 minutes .
Heat inactivate at 65 degrees C for 10 minutes .
Chill on ice and transform 1 - 5 μl of the reaction into 50 μl competent cells .
