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From 2012.igem.org

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Revision as of 23:40, 3 October 2012

I. Check Transformation Plates for Colonies

RESULTS:

• B. subtilis PY79 - no colonies on any plates

• E. coli MachI

Plate	colonies
- control	0
+ control (pBAV1K)	~4000 white
1	~0 white, ~1500 blue
2	~1500 colonies, 30-50% blue
3	~0 white, ~300 blue
4	1-3 white, 300-400 blue

• A. baylyi ADP1

Plate	colonies
- control	0
+ control (pBAV1K)	>10000 white
1	14 white, 0 blue
2	11 white, 0 blue
3	1 white
4	23 blue, 12 white (note, white colonies were significantly larger than all except two blue colonies)

II. Make liquid cultures from Transformation Plates

• Aliquot 1 mL of LB each to 3 tubes
  • To all three, add 1 uL of Kan stock (50 mg/mL)
  • To one, add 1 uL of Chloramphenicol stock (37 mg/mL)
  • In the dark, to another, add 1.5 uL of Tetracycline stock (10 mg/mL)
• Repeat for each plate tested

• Inoculate one blue colony (except for WT plates) into one tube according to the following list:
  • ADP1 WT (Kan, Kan+Cm, Kan+Tet)
  • ADP1 4 (Kan, Kan+Cm, Kan+Tet)
  • MachI WT (Kan, Kan+Cm, Kan+Tet)
  • MachI 1 (Kan, Kan+Cm, Kan+Tet)
  • MachI 2 (Kan, Kan+Cm, Kan+Tet)
  • MachI 3 (Kan, Kan+Cm, Kan+Tet)
  • MachI 4 (Kan, Kan+Cm, Kan+Tet)
    • Note: because of the small number of large blue colonies on the ADP1 4 plate, I had to hit the same colony several times

• Incubate overnight with shaking at 32 ^oC