IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Output/2009/06/29/Zhangs
1. Double digestion for Terminator vector again causing the digestion product’s been exhausted.
(1) Got 2×100μL DH5α competent cells from the -70℃ refrigerator and divided them equally into four EP tubes, each for 50μL.
(2) Wait for the solution to melt.
(3) Add 10μL ligation system: 1-23L 7:1, 1-23L 3:1, 1-4H 7:1, 1-4H 3:1.
Notice: 7:1, 3:1 are the ratio of insert (CI) to vector (Terminator).
(4) Keep in ice for 30min.
(5) Heat shock in 42℃ for 90s.
(6) Put the competent cells back into the ice box immediately for 2min.
(7) Add 200uL antibiotic LB in a super clean bench and recover for 45min.