General Information
| Overview |
T4 DNA ligase catalyzes the formation of a phosphodiester bond between 5' terminal phosphate and adjacent 3' hydroxyl termini in duplex DNA, RNA or DNA/RNA hybrids. This enzyme will join blunt end and cohesive end termini but it cannot repair single stranded nicks. |
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| Protocol |
1. Prepare the ligation reaction mixture in a microcentrifuge tube.
2. Incubate the reaction mixture at 16°C overnight.
3. Transformation 1. Take the competent cells out of the -80°C refrigerator, and place the competent cells immediately in an ice water bath. 2. Add the DNA into 100 μl of competent cells and mix gently. Keep in the ice for 30 minutes. 3. Incubate the mixture at 42°C for 90 seconds. And then return to the ice bath for 2~3 minutes. 4. Add 900 μl SOC or LB medium and culture by shaking (150 rpm) at 37°C for 45 minutes to recovery. 5. Centrifuged at 2500 xg for 5 minutes, and then remove 900 μl of the supernatant. Mix the remaining solution and plate on selective media. 6. Incubate at 37°C overnight. |
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