Consort EVS1000 Series Manual - Página 8
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Protein (Western) blotting buffers
Transfer buffers must be made accurately using high grade reagents. Do not adjust the pH
with acid or base as this will affect the properties of the buffer. pH will vary according to the
purity of the reagents used.
Triple buffer system
For high efficiency transfer of Protein from acrylamide gels
•
Anode 1 Buffer:
0.3 M Tris base, 20% MeOH, pH 10.4:
soak 4 standard grade filter paper sheets
•
Anode 2 Buffer:
0.025 M Tris base, 20% MeOH pH 10.4:
soak 2 standard grade filter paper sheets
•
Cathode Buffer:
0.025 M Tris base, 0.04 M Caproic Acid, 20% MeOH pH 9.4:
soak 6 standard grade filter paper sheets
DNA (Southern) Blotting Buffer
For high efficiency transfer of DNA from agarose gels.
•
Soak 12 pieces of standard grade filter paper, 6 for the cathode, 6 for the anode in:
o
50 x 1 M ethanolamine-glycine buffer, pH 11
RNA (Northern) Blotting Buffer
For high efficiency transfer of RNA from agarose gels.
•
Soak 12 pieces of standard grade filter paper, 6 for the cathode, 6 for the anode in:
o
50 x 0.2 M morpholinopropanesulfonic acid (MOPS)
o
50 mM sodium acetate
o
5 mM EDTA pH 7.0
Nucleic acid transfer buffers
Soak 12 pieces of standard grade filter paper, 6 for the cathode, 6 for the anode in:
•
1 x TAE 40 mM Tris (pH 7.6), 20 mM acetic acid, 1 mM EDTA
•
50 x (1l) dissolve in 750 ml distilled water:
o
242 g Tris base (FW = 121)
o
57.1 ml glacial acetic acid
o
100 ml 0.5 M EDTA (pH 8.0)
•
Fill to 1 litre with distilled water.
Soak 12 pieces of standard grade filter paper, 6 for the cathode, 6 for the anode in:
•
1 x TBE 89 mM Tris (pH 7.6), 89 mM boric acid, 2 mM EDTA
•
10 x (1l) dissolve in 750 ml distilled water:
o
108 g Tris base (FW = 121)
o
55 g boric acid (FW = 61.8)
o
40 ml 0.5 M EDTA (pH 8.0)
•
Fill to 1 litre with distilled water.
Transfer buffers
Towbin Buffer with 20% methanol
Soak 12 pieces of standard grade filter paper, 6 for the cathode, 6 for the anode in:
•
0.025 M Tris base
•
0.192 M glycine
•
20% methanol
•
pH 8.3
EVS1000 Blot units
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