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

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(Sample preparation)
(Sample preparation)
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 (suspended in PBS)
+
#* 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 11:54, 26 October 2006

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
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