"

From 2012.igem.org

Contents 1 I. Re-check plates that were allowed to grow more overnight 1.1 RESULTS: 2 II. Check Overnight Liquid Cultures 2.1 RESULTS: 3 III. Re-make liquid cultures from Transformation Plates

I. Re-check plates that were allowed to grow more overnight

RESULTS:

No appreciable change in E. coli plates. 2 blue colonies developed on the ADP1 1 plate and 5 more blue colonies developed on the ADP1 4 plate. Blue colonies had grown larger.

II. Check Overnight Liquid Cultures

RESULTS:

It was unclear which cultures had grown, if any. Most of the positive control tubes (kanamycin alone) have not grown. I will repeat the experiment with IPTG as well.

III. Re-make liquid cultures from Transformation Plates

• Media
  • Aliquot 12 mL each of LB media to 2 15 mL conical tubes
  • To each, add 12 uL of Kan stock (50 mg/mL), mix
  • In the dark, to one of the 12 mL preparations, remove 60 uL of solution and replace with 60 uL of IPTG stock (0.1 M) for 0.5 mM solution
  • For each preparation, aliquot 4 mL each to two other 15 mL conical tubes
    • To one, add 4 uL of Chloramphenicol stock (37 mg/mL)
    • To the other, add 6 uL of Tetracycline stock (10 mg/mL)
  • Aliquot all preparations such that a 5 mL culture tube gets 0.5 mL of only one of the solutions and label accordingly

• Inoculate one blue colony (except for WT plates) into one tube according to the following list:
  • ADP1 WT (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • ADP1 1 (Kan, Kan+Tet) <-- only two blue colonies
  • ADP1 4 (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • MachI WT (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • MachI 1 (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • MachI 2 (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • MachI 3 (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)
  • MachI 4 (Kan, Kan+Cm, Kan+Tet, IPTG+Kan, IPTG+Kan+Cm, IPTG+Kan+Tet)

• Incubate overnight with shaking at 32 ^oC