User:Catherine Koenigsknecht/Notebook/Experimental Biological Chemistry/2011/10/25: Difference between revisions

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==Description==
==Description==
#  
This procedure uses the Bio Rad Mini Protean system:
 
# Make the resolving gel:
#* 3.3mL H<sub>2</sub>O
#* 4.0mL 30% acrylamide mix
#* 2.5mL 1.5M Tris (pH 8.8)
#* 0.1mL 10% SDS
#* 0.1mL 10% ammonium persulfate (add this right before pouring the gel)
#* 0.004mL TEMED (add this right before pouring the gel)
# Pour the gel between the short plate and spacer plate (after they have been secured in the casting frame on the casting stand). Squirt a layer of methanol on top of the resolving gel for a straight, level layer of gel.
# Remove the methanol with chromatography paper.
# Make the stacking gel:
#* 3.4mL H<sub>2</sub>O
#* 0.83mL 30% acrylamide mix
#* 0.63mL 1.0M Tris (pH 6.8)
#* 0.05mL 10% SDS
#* 0.05mL 10% ammonium persulfate
#* 0.005mL TEMED
# Pour this layer of gel on top of the resolving layer between the two glass plates. Put in a 10-well comb. Wait until it is polymerized.
# Remove the comb and transfer the gel within the plates to the Electrode Assembly with the short plates facing inward.
# Fill the Electrophoresis chamber with Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)
# Load 10μL of pre-mixed SDS-PAGE broad range ladder.
# Load 16μL of each protein sample mixed with 4μL of 5x loading buffer (pre-mixed) into the wells.


==Data==
==Data==

Revision as of 09:43, 26 December 2011

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Entry title

Objective

Learn how to maintain an OpenWetWare Notebook.

Description

This procedure uses the Bio Rad Mini Protean system:

  1. Make the resolving gel:
    • 3.3mL H2O
    • 4.0mL 30% acrylamide mix
    • 2.5mL 1.5M Tris (pH 8.8)
    • 0.1mL 10% SDS
    • 0.1mL 10% ammonium persulfate (add this right before pouring the gel)
    • 0.004mL TEMED (add this right before pouring the gel)
  2. Pour the gel between the short plate and spacer plate (after they have been secured in the casting frame on the casting stand). Squirt a layer of methanol on top of the resolving gel for a straight, level layer of gel.
  3. Remove the methanol with chromatography paper.
  4. Make the stacking gel:
    • 3.4mL H2O
    • 0.83mL 30% acrylamide mix
    • 0.63mL 1.0M Tris (pH 6.8)
    • 0.05mL 10% SDS
    • 0.05mL 10% ammonium persulfate
    • 0.005mL TEMED
  5. Pour this layer of gel on top of the resolving layer between the two glass plates. Put in a 10-well comb. Wait until it is polymerized.
  6. Remove the comb and transfer the gel within the plates to the Electrode Assembly with the short plates facing inward.
  7. Fill the Electrophoresis chamber with Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)
  8. Load 10μL of pre-mixed SDS-PAGE broad range ladder.
  9. Load 16μL of each protein sample mixed with 4μL of 5x loading buffer (pre-mixed) into the wells.

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.



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