IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-26: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
JeffreyLau (talk | contribs) |
JeffreyLau (talk | contribs) |
||
| Line 22: | Line 22: | ||
# Resuspend pellet in 100 uL PBS. You can freeze this for future Western blots if you wish. | # Resuspend pellet in 100 uL PBS. You can freeze this for future Western blots if you wish. | ||
# Make a 20 uL mixture containing the following: | # Make a 20 uL mixture containing the following: | ||
#* 5 uL sample | #* 5 uL PBS-suspended sample | ||
#* 11 uL H<sub>2</sub>O | #* 11 uL H<sub>2</sub>O | ||
#* 4 uL 5x sample buffer | #* 4 uL 5x sample buffer | ||
Revision as of 08:54, 26 October 2006
Western blot
DUN DUN DUN
Lanes
- SeeBlue Plus2 marker
- J36010 (Promoter + RBS + KaiC) @ 0.55 OD
- J36021 (Promoter + RBS + KaiB + Promoter + RBS + KaiC) @ 0.55 OD
- J36022 (Promoter + RBS + KaiC + Promoter + RBS + KaiC) @ 0.55 OD
- J36010 @ 0.4 OD
- J36021 @ 0.4 OD
- J36022 @ 0.4 OD
- J36010 @ 0.18 OD
- J36021 @ 0.18 OD
- J36022 @ 0.18 OD
- GFP dev (positive control) @ 0.4 OD
- SeeBlue Plus2 marker
Sample preparation
At Alain's recommendation, I prepared the samples differently from the procedure in the Endy protocol.
- Spin down 1 mL of culture at 13K for 2 minutes, freeze pellet
- Resuspend pellet in 100 uL PBS. You can freeze this for future Western blots if you wish.
- Make a 20 uL mixture containing the following:
- 5 uL PBS-suspended sample
- 11 uL H2O
- 4 uL 5x sample buffer
- Boil for 2 minutes
- Spin down briefly to get condensation off the top of the tube
- Insert into lanes
