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The Milan protocol ...: Difference between revisions
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* Take 3 μg of the vector and 3 μg of the insert and treat separately with 0.5 U of T4 DNA polymerase (1 μl) in T4 buffer (buffer 2 NEB) plus BSA in a 60 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice. Scale reagents according to your needs. | * Take 3 μg of the vector and 3 μg of the insert and treat separately with 0.5 U of T4 DNA polymerase (1 μl) in T4 buffer (buffer 2 NEB) plus BSA in a 60 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice. Scale reagents according to your needs. | ||
* Set up a 20 μl annealing reaction using 1:1 insert to vector ratio with 500 ng of a (5 kb) vector, 1x T4 DNA ligation buffer (NEB), appropriate amount of insert, and water. Incubate at room temperature for 30 minutes. Leave on ice or store in - | * Set up a 20 μl annealing reaction using 1:1 insert to vector ratio with 500 ng of a (5 kb) vector, 1x T4 DNA ligation buffer (NEB), appropriate amount of insert, and water. Incubate at room temperature for 30 minutes. Leave on ice or store in -20°C. | ||
* Add 10 μl of the annealed mixture to 100 μl of TOP10 chemical competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, return to ice for 2 minutes, add 0,9 ml of LB, recover at 37°C for 1 hour. | * Add 10 μl of the annealed mixture to 100 μl of TOP10 chemical competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, return to ice for 2 minutes, add 0,9 ml of LB, recover at 37°C for 1 hour. | ||