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Ligation Attempt #2 continued

  • Following digestion and purification, took concentrations of digestion products, which were still miserably low (phsABC: 3.2 ng/uL, P0312: 16.88 ng/uL, J23114: 23.98 ng/uL, B0015: 13.24 ng/uL) and set-up ligation attempt #2. For each of the two ligations (phsABC into B0015 and J23114 into P0312) four different 20 uL reactions were run: a control ligation with vector, ligase and buffer but no insert, a reaction with a 2:1 stoichiometric ratio of insert to vector, a reaction with a 3:1 insert to vector ratio, and a reaction with a 5:1 insert to vector ratio. All reactions used 1 uL NEB T4 ligase and 2 uL of accompanying 10x buffer and were run for 15 minutes at room temperature. Following this the (theoretically) ligated plasmids were transformed into competent XL1-Blue cells according to the standard transformation protocol and plated onto Amp plates.
  • Index plates from ligation attempt #1 on 7/1 belatedly showed growth.

This day's activities are also recorded on page 45 of the hard copy lab notebook.