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 samples differently from the procedure in the Endy protocol.

1. Spin down 1 mL of culture at 13K for 2 minutes, 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 PBS-suspended sample
   • 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

Gel preparation

Remove the white tape from the gel. Notice the horizontal line under the white tape? Later you will run the gel until the blue stain frontier reaches this line.

Flush the wells with water.

Mark the bottom of each well with a sharpie. This is a useful guide for your pipette when you insert your samples.

Running the gel

Run at 125V until the blue frontier reaches the bottom horizontal line that was under the white tape when you opened the gel package.