T4 DNA Ligation Protocol

[domba]

Supplies:

1. Microcentrifuge and adaptors for 600 ul tubes
2. Micropipetter and tips

Procedures:

1. Set up the following reactions: (total of 10 ul)

Sample Control
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distilled water 6.5 ul 7 ul
10 X ligase reaction buffer 1 ul 1 ul
vector (at a concentration of 0.5-2 ug/ul) 1 ul 1 ul
insert (at a concentration of 3-5 ug/ul) 1 ul 1 ul
T4 DNA ligase 0.5 ul 0 ul

2. Mix gently and spin for 2 seconds in a microcentrifuge.
3. Incubate at 4 degrees C or 20 degrees C (room temperature) overnight.

Results:

1. In the sample tube: insert is ligated with vector to form circular plasmid DNA. In the control tube: vector and insert remain separate without T4 DNA ligase.