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