Registry/Measurement kit/Notebook/2007-7-25

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  • F2620 was ligated into 3K3(E/P) instead of E/X
  • Re-ligated F2620 (M/X) into 3K3 (E/X) and 1AK3 (E/X)
    • Reaction with 3K3 was left in incubator too long. Made another reaction, will transform both.
  • Transformations went into warm room at 2:30
  • Noticed that F2620 has an internal MfeI site. This could cause problems later on when re-ligating.
    • Could not find alternative enzyme on NEB's site. (ApoI cuts R'AATT_Y, thus is an isoschizomer of EcoRI [G'AATT_C]. Since there is an EcoRI site on the reverse tail, we can't use this.)


  • Also Digesting more 3K3 (E/P) Making o/n of 3K3
  • Mini-prepped 1AK3, digested E/X, E/P


  • Ligating into 1AK3; ran out of 3K3.


  • Re-trying promoter PCR

I2055 Synthesis

  • Making (hopefully silent) 1bp change to remove internal MfeI site. (CAATTG -> CAATCG [codon ATT->ATC (Ile)])


  • Started PCRs with E/X tails and S/X tails.

Transformations/overnight stuff

  • O/n of 3K3
  • Transformation of F2620-3K3 (2 samples), F2620-1AK3, and I2056-1AK3.
  • Digesting 1AK3 E/X, E/P