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User:DrJones1935/30 September 2012
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Contents |
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 |
Close-up of blue colonies
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
