C.B.S.
Scientific Co, Inc.
9
Semi-Dry
Blotters
a. Transfer DNA of agarose gels (Southern) requires some special precautions.
Excessive heat build-up can melt agarose if too much power is applied. To
avoid this, try to use higher percentage gels made from high melt agarose.
b. With this type of agarose, run at low current (100-200mA for 15-30 minutes,
depending on the gel thickness.
c. If you are using low melt agarose or low percentage agarose, use 50mA for
45-60 minutes.
5. Single stranded DNA from 6% Acrylamide
a.
100-125mA @ constant current for 45-60 minutes (0.5-1XTBE)
B. Semi-Dry Blotting of Proteins (Western)
1. Preparation for Western Blotting
a. Acrylamide gels 20cm x 20cm and up to 3mm thick may be transferred in
these systems.
b. Buffer
System
192mM
Glycine Towbin
reference
25mM Tris, pH 8.3
0.0013M SDS
10-20% MeOH
39mM
Glycine
Maniatis
reference
48mM Tris, pH 8.3
0.0375% (w/v) SDS
20% (v/v) Methanol
c. If the gel is to be stained with Coomasie Blue prior to blotting, refer to Perides,
et. al. (1986) for an alternate protocol before blotting.
2. Prior to completion of SDS-PAA electrophoresis:
a. Prepare a Mylar™ mask by cutting a rectangular hole that is slightly smaller
(1-2mm) than the gel dimensions.
b. Prepare 8 pieces of Whatman 3mm chromatography paper the exact size of
the gel. Larger pieces of filter paper may promote a ‘short circuit’ by allowing
current to bypass the gel and therefore reduce the efficiency of the transfer.
Soak the filter paper in transfer buffer until saturated. The ‘ready for use’
thickness should be around 1mm (dry paper is approximately .35mm).
c. Hydrate the Nitrocellulose by first floating in a tray of de-ionized water
(capillary absorption) followed by 5 minutes of total immersion.
3.
Stacking of components into Electro-Blotter Apparatus
a. After SDS-PAA electrophoresis of proteins, separate the gel plate sandwich
and briefly soak the gel in a tray of de-ionized water. Remove the stacking
gel with a spatula or razor blade and discard.
b. Wet the anode and cathode plates by wiping with a lint free paper towel. No
puddles should be left in the unit.
c. Insert the Mylar mask over the bottom platinum coated titanium electrode
(anode +).
Note:
The bottom electrode is the anode and is fixed in place,
while the cathode is adjustable and within the lid of the apparatus. Place 3-4
pieces of saturated filter paper carefully centered over the cutout. Flatten the
filter paper by rolling a glass rod over the surface.
