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

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(Western blot)
Line 2: Line 2:
DUN DUN DUN
DUN DUN DUN
-
Lanes:
+
=== Lanes ===
# SeeBlue Plus2 Standard marker
# SeeBlue Plus2 Standard marker
# J36010 (Promoter + RBS + KaiC) @ 0.55 OD  
# J36010 (Promoter + RBS + KaiC) @ 0.55 OD  
Line 15: Line 15:
# GFP dev (positive control) @ 0.4 OD
# GFP dev (positive control) @ 0.4 OD
# SeeBlue Plus2 Standard marker
# SeeBlue Plus2 Standard marker
 +
 +
=== Sample preparation ===
 +
At Alain's recommendation, I prepared the sample differently from the procedure in the [[IGEM:Harvard/2006/Cyanobacteria/Western Blot|Endy protocol]].
 +
 +
# Spin down 1 mL of culture, 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 sample (suspended in PBS)
 +
#* 11 uL H<sub>2</sub>O
 +
#* 4 uL 5x sample buffer
 +
# Boil for 2 minutes
 +
# Spin down briefly to get condensation off the top of the tube
 +
# Insert into lanes

Revision as of 11:53, 26 October 2006

Western blot

DUN DUN DUN

Lanes

  1. SeeBlue Plus2 Standard 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 Standard 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
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