IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-26

Western blot

DUN DUN DUN

Lanes

1. SeeBlue Plus2 marker
2. J36010 (Promoter + RBS + KaiC) @ 0.55 OD
3. J36021 (Promoter + RBS + KaiB + Promoter + RBS + KaiC) @ 0.55 OD
4. J36022 (Promoter + RBS + KaiC + Promoter + RBS + KaiC) @ 0.55 OD
5. J36010 @ 0.4 OD
6. J36021 @ 0.4 OD
7. J36022 @ 0.4 OD
8. J36010 @ 0.18 OD
9. J36021 @ 0.18 OD
10. J36022 @ 0.18 OD
11. GFP dev (positive control) @ 0.4 OD
12. SeeBlue Plus2 marker

Sample preparation

At Alain's recommendation, I prepared the sample differently from the procedure in the Endy protocol.

1. Spin down 1 mL of culture, freeze pellet
2. Resuspend pellet in 100 uL PBS. You can freeze this for future Western blots if you wish.
3. Make a 20 uL mixture containing the following:
   • 5 uL sample (suspended in PBS)
   • 11 uL H₂O
   • 4 uL 5x sample buffer
4. Boil for 2 minutes
5. Spin down briefly to get condensation off the top of the tube
6. Insert into lanes