• Morning: We carried out several digestion:
  • GFP with E/P in buffer 2 for 1 hour; in buffer 2 for 2 hours; and in EcoRI buffer for 2 hours (this aims to compare the efficiency of buffer 2 with EcoRIbuffer for E/P double digestion; as well as the optimal digestion time).
  • GFP with X/P in buffer 3 for 2 hours.
  • E7 with X/P in buffer 3 for 2 hours.
  • E7 and T7ptag with E/P in buffer 2 for 1.5 hours.
    For all the digestions, the amounts used are:

DNA: 1000ng (around 5ul)
Enzyme1: 1 ul
Enzyme2: 1 ul
Buffer: 5 ul
BSA: 0.5 ul
H2O: 37.5 ul
Total: 50

• 1240: MinElute PCR pufification for the above 7 mixtures (obtain 10ul each after purification).
• 1650: gel extraction. After this, the digested DNAs were put in -20 fridge for ligation on Thursday.
• 1520: digestion for gel check of 12 samples of Fe-GFP inoculated yesterday. However,no colony was found correct.