Wet the two pieces of thick blotting paper in the appropriate transfer buffer an

d place them
on top of the wet membrane. Roll a pipette across the surface of the membrane to
smooth
away any air bubbles.
16. Cut or fold a stack of paper towels (5-8 cm high) just smaller than the blot
ting papers. Place
the towels on the blotting papers. Put a glass plate on top of the stack and wei
gh it down with
a 400-g weight.
The objective is to set up a flow of liquid from the reservoir through the gel a
nd the membrane so
that fragments of denatured DNA are eluted from the gel and are deposited on the
membrane. The
weight on the top of the gel should be heavy enough to ensure good contact betwe
en the various
components of the blot, but light enough to prevent compressing the gel. Compres
sion will
squeeze liquid from the interstices of the gel, leaving a dehydrated matrix that
greatly retards the
movement of DNA and drastically reduces the efficiency of transfer from the gel
to the membrane.
Intact, rather than cut or folded, paper towels can also be used in this setup,
but only if the protective mask of Saran Wrap or Parafilm efficiently prevents s
eepage of buffer.
To prevent evaporation, some investigators wrap the entire transfer setup in Sar
an Wrap. Whether
this is necessary seems somewhat doubtful.
17. Allow the transfer of DNA to proceed for 8-24 hours. Replace the paper towel
s as they
become wet. Try to prevent the entire stack of towels from becoming wet with buf
fer.
18. Remove the paper towels and the blotting papers above the gel. Turn the gel
and the attached
membrane over and lay them, gel side up, on a dry sheet of blotting paper. Mark
the positions of the gel slots on the membrane with a very soft lead pencil or a
ballpoint pen.
19. Peel the gel from the membrane and discard the gel.
Instead of discarding the gel, it can be stained (45 minutes) in a 0.5 flg/ml so
lution of ethidium
bromide in H20 and visualized on a UV transilluminator to gauge the success of t
he DNA transfer. Note that the intensity of fluorescence will be quite low becau
se any DNA remaining in the gel
will have been denatured.
Fixation of the DNA to the Membrane
The sequence of steps from immobilization of DNA to the membrane to subsequent h
ybridization depends on the type of membrane, the method of transfer, and the me
thod of fixation
(please see Table 6-3). Because alkaline transfer results in covalent attachment
of DNA to positively charged nylon membranes, there is no need to fix the DNA t
o the membrane before
hybridization. DNA transferred to uncharged nylon membranes in neutral transfer
buffer should
be fixed to the membrane by baking under vacuum or heating in a microwave oven,
or crosslinked to the membrane by UV irradiation.