Equipment
- - Two glass plates
- - Gel caster
- - Comb
- - VWR Power Source 300V
Chemicals & consumables
- - SDS
- - Rotiphorese® (30%)
- - Tris HCl
- - Glycine
- - TEMED
- - APS
- - Aqua dest.
- - Isopropyle alcohol
- - Glycerine
- - Beta-Mercaptoethanol
- - Bromphenolblue
Buffers & gels
- - Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)
- - Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)
- - Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)
- - Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))
- - Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)
- - 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH = 6.75)
Procedure
- prepare the separating gel and fill it into the chamber
- pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
- discard the isoprpyl alcohol and pour the prepared stacking gel
- stick in the comb
- if not used, store the gel in wet cloth (to prevent dehydration) at 4°C
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