Pharmacia Product Number: 27-0361-01
1. DNA used for ligation can be in water or TE buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA).
2. Final volume of ligation must be 20ul. Otherwise, add RNase-free water to bring the volume up to 20ul.
3. Incubate the reaction at room temperature for 5 minutes.
4. Mix the reaction by pipetting up and down a couple times.
5. Centrifuge the tube at 14K rpm for 1 minutes to bring the contents to the bottom of the tube and remove bubbles created by pipetting.
6. Incubate the reaction at 16oC for 30 (cohesive-ended DNA) to 45 (blunt-ended DNA) minutes.
7. The ligase can be inactivated by heating at 70oC for 10 minutes.
8. Use 2ul of the ligation reaction for transformation of 100ul of competent cells. Larger volume than 2ul may affect transformation efficiency due to the stabilizer present in the reaction. If more than 2ul is required for transformation or electroporation is required, then salt precipitate the DNA as followed:
DNA Salt Precipitation
Add 5-13ul of 5M NaCl stock solution to make the ligation reaction NaCl concentration 1-2M.
Add 2 volumes of RT 100% EtOH.
Vortex tube and place it at -70oC for at least 1 hr.
Centrifuge the tube at max speed for 10 minutes.
Carefully discard the supernatant.
Wash the pellet twice with 1mL of ice cold 70% EtOH. Centrifuge at max for 5 min & discard supernatant carefully each time.
Dry the pellet by placing the tube in the 37oC incubator for about 15 minutes.
Resuspend the pellet in water or TE buffer.