C.B.S. Scientific EBU-6000 - Manual (Page 11)

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3. Preparation of the Apparatus
a. Prepare 10 sheets of blotting paper cut to the same size of the gel and soak in
0.1TXAE. Cut a nylon membrane to the same size and wet in ddH
2
O for 5
minutes followed by equilibrate in 0.1XTAE for 20-30 minutes.
b. Prepare one of the clear plastic Mylar sheets (provided in the kit) by cutting a
hole in the sheet which is 1-2mm smaller than the gel dimension.
c. Insert the Mylar mask over the bottom electrode anode. Place 5 pieces of
saturated filter paper carefully centered over the cut-out in the Mylar mask. If
prepared correctly, the filter paper will overlap the mask cut-out slightly.
Flatten the filter paper by rolling a glass rod over the surface. Apply the
equilibrated Nylon membrane to the stack of filter paper followed by the gel
and 5 more pieces of saturated filter paper. Again roll a glass rod over the
filter paper stack to compress and remove trapped air bubbles. 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. Electro-blotting transfer
a. Once the lid is in place, insert the power cords into the apparatus. Plug the
ends of the power cords into the Power Supply.
b. Set the Power Supply at 250mA and transfer for 30 minutes at constant
current.
c. After the run, turn off the power supply and disconnect the power cords from
the apparatus. The lid cannot be lifted unless the power cord is unplugged
from the anode. Carefully lift the lid and watch for filter paper sticking to the
top electrode. Remove the Nylon membrane and process according to
protocol. Rinse the inside of the apparatus and the mask with ddH
2
0.