Gel Sandwich Assembly - Thermo Scientific OWL HEP-1 Operating And Maintenance Manual

Section 2
Setting Up
DNA Gels (continued)
Gel Sandwich
Assembly
2-4 Semidry Electroblotter
5. An alternative procedure at this step is to peel the paper, with the gel
attached, from the glass plate upon which it rests. Beginning at one
end of the gel, slowly lift and peel back the paper with the gel attached.
As before, a stream of water from a wash bottle can be sprayed at any
spot to aid separation of the gel from the glass plate.
6. Place the gel resting on the Whatman, 3MM paper flat on a lab bench.
7. Wearing gloves, cut a piece of nylon membrane the size of the gel and
blotting paper. Briefly (but thoroughly) wet the membrane in 0.5 x
TBE in a shallow tray, being careful not to crease the membrane.
Holding the membrane by two corners, allow the excess liquid to drip
from the membrane into the tray.
1. Wearing gloves, cut the membrane to the size of the gel and blotting
paper.
2, Mark the membrane, to indicate the side to which the samples will be
on. This is important in the event that any successive probe is negative,
and to indicate sample orientation. This can be done by either clipping
a comer of the membrane or using a ball point pen. Clip the same
comer until you retire.
3. Wet the membrane according to its manufactures recommendations,
followed by a quick equilibration in transfer buffer. It is often helpful
to have all the filter paper and membranes sitting in transfer buffer as
you start to build the blotting sandwich.
4. With this device, the transfer is in the upward direction as shown in
the figure below, and the blotting sandwich is built upward, upon the
mirrored stainless steel plate of the bottom half of the blotter.
5. Lay three filter pads, one after the other down. Each should be soaking
wet with buffer. In fact, the lower chamber may filled with buffer as
you build the blotting sandwich.
6. Add a few mL of buffer to the filter pad, and gently layer the gel.
Beginning at one end of the filter paper, align the gel with the paper's
edge, and slowly lower the other end, driving out any bubble.
7. Wearing gloves, gently smooth out any bubbles by forcing them to the
closest edge of the gel. Test tubes and pipettes many also be used for
this purpose.
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