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 titleObjectiveLearn how to maintain an OpenWetWare Notebook. DescriptionThis procedure uses the Bio Rad Mini Protean system:
Data
NotesThis area is for any observations or conclusions that you would like to note.
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