User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/29

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11/29/09

  • ✓ Cultures: KAH99(2), 100(2), 101(2), 102(2), 103(2), 104(2)
  • ✓ Oligo annealing: KAH012 (phosphorylate, anneal)

Oligo annealing

  1. KAH012

---> Phosphorylate oligos, then anneal overnight

DNA oligos (100 μM) up to 21 μL (3 μL ea.)
10x PNK buffer (NEB) 3.0
ATP (10 mM) 2.0
T4 PNK (NEB) 2.0
dH2O 2.0
  30 μL

--> 37°C/ 1 hr.
--> Add 4 μL 500 mM NaCl
--> 100°C waterbath/ 10 min.
--> Cool to R.T. overnight