C.B.S. Scientific EBU-6000 - Manual (Page 8)

www.cbsscientific.com 8 Semi-Dry Blotter Manual 12/12
B. Semi-Dry Electro-Blotting of DS DNA from Agarose Gels (Southern)
1.
Preparation for Southern Blotting
a. Agarose gels up to 6mm thick may be run in these systems. Thicker gels
require longer transfer times. Prior to transfer, gels should be stained
with Ethidium Bromide. Ethidium bromide is a Mutagen. ALWAYS wear
gloves. Dispose of solutions in accordance with the safety regulations of
your institution.
b. Optionally, depurinate the gel by soaking in 0.25M HCl for 20 minutes.
Depurination can increase transfer efficiency. To denature the DNA in
the gel before transfer, soak in 0.5M NaOH, 1.0M NaCl for 20 minutes.
Neutralize 2X for 15 minutes in 0.5M Tris-HCl, pH 7.4, 1.5M NaCl.
c. Membrane selection: Nylon or nylon supported nitrocellulose. ALWAYS
use forceps to handle membranes. If nylon is not base resistant,
denature DNA prior to transfer. If it is, you may base wash the DNA after
transfer instead of denaturing before. Base wash for 30 min. in 0.1N
NaOH followed by suspension in 0.2M Tris-base, pH 8.0, 0.5% SDS for
30 minutes to neutralize the membrane.
2. Preparation of the Transfer Materials
a. Required Materials: Membrane, cut to exact size of gel; saturate in ddH
2
O
for ten minutes, then in 0.1X TAE or 0.3X TBE for 20 minutes.
b. Gels should be soaked in running buffer, 0.1XTAE or 0.3XTBE for 20-60
minutes depending on thickness (3-6mm).
c. Cut eight sheets of 3mm blotter paper (Whatman) the exact size of the
gel. Saturate all eight sheets in running buffer.
d. Mylar Mask: Prepare a mask by cutting a rectangular hole that is slightly
smaller (1-2mm) than the gel dimensions.
e. For transfer of ssDNA (acrylamide): use 0.5 –1X TBE buffer. Equilibrate
in transfer buffer for 15 minutes.
3. Stacking of components into Electro-Blotter Apparatus:
a. 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 cut-out.
Flatten the filter paper by rolling a glass rod over the surface.
b. Apply a saturated membrane on the top of the paper.
c. Carefully place the gel on the membrane, making sure no bubbles remain
between the gel/membrane interface.
d. Stack the remaining 3-4 sheets of blotting paper on the top of the gel.
Add one sheet at a time and be sure to ‘smooth’ with glass rod to remove
air bubbles.
e. Lift the cathode assembly using the white handle in the center of lid; place
lid directly over the stack and apply downward pressure while tightening
each of the nylon thumb screws.
4. Power Setting
Connect the unit to the power supply using the power cords supplied. Warning:
C.B.S. Scientific has designed this device such that it cannot be opened unless
the power cords are disconnected. DO NOT attempt to open the transfer
apparatus without disconnecting the power cords and shutting off the power
supply.