Bio-Rad Zeta-Probe Membranes - Manual (Page 5)

7. Cut two pieces of 3MM exactly to the gel size. Wet a sheet of
precut 3MM paper in water and place it on the Zeta-Probe
membrane/gel stack, then repeat with the second sheet.
Remove any bubbles from beneath each layer of 3MM paper.
8. Place a stack of precut paper towels on the 3MM/Zeta-Probe
membrane/gel stack. Cover the paper towel stack with a plastic or
glass plate. Keep the pressure on the paper towel stack at a
minimum. Excessive weight will compress the gel, retarding
capillary transfer.
9. Continue transferring for 2–24 hours, depending on the gel
concentration and fragment size. Note: Higher background may
appear if transfer is longer than 24 hr.
10. After transfer, remove the stack of paper towels. Gently peel the
Zeta-Probe membrane from the surface of the gel, rinse it in
2x SSC, and air dry. DNA is fixed to the membrane during
transfer, eliminating the need for subsequent fixation. The dried
membranes are stable at room temperature. The membranes
can be stored dry between two pieces of filter paper in plastic
bags at 23–25°C.
2.4 Electrophoretic Transfer
The following protocol was developed for maximum efficiency of
electrophoretic transfer. It affords the greatest mobility of DNA and
RNA, and the most complete transfer from gel to membrane without
excessive heat generation. The buffer (ionic strength and pH) and field
strength have been optimized for electrophoretic blotting of DNA and
RNA from both agarose and acrylamide gels. For electrophoretic
transfer from agarose gels, a heat exchanger must be used, because
increased temperatures could melt the agarose gel. The protocol was
developed using the Trans-Blot
® electrophoretic transfer system with a
heat exchanger.
5
pour 95°C 20 mM Tris-HCl, pH 8.0, 1 mM EDTA onto the blotted
membrane, then gently agitate at room temperature until the solution
cools. After removal of glyoxal adducts, proceed to hybridization or
store the membranes dry.
2.3 Alkaline Blotting 3 (DNA Capillary Transfer)
1. Depurinate the DNA by soaking the gel in 0.25 M HCl for
10–15 min. Rinse the gel several times with distilled water.
Note: Acid depurination is only recommended for fragments
>4 kb.
2. Cut four sheets of Whatman 3MM paper so they overhang the
bottom of the gel tray by 5 cm on each end. Prewet the Zeta-
Probe membrane in distilled water.
3. Place the four sheets of 3MM paper on an inverted gel casting
tray. Place the 3MM/tray in the bottom of a deep dish. Then sat-
urate the 3MM paper with 0.4 M NaOH. Remove the bubbles by
repeatedly rolling a glass pipet over the saturated 3MM paper.
Pour enough NaOH into the deep dish so that the 3MM wick
ends are immersed in NaOH.
4. Pour more NaOH onto the 3MM wick to saturate it, then carefully
place the gel on the wick. Make sure that no bubbles are trapped
beneath the gel. Cover the gel with a small amount of NaOH.
5. Place plastic wrap (such as Saran wrap) over the entire gel/3MM
stack. Cut out a window with a clean razor blade, allowing only
the gel to be exposed.
6. Lower the sheet of pre-wetted Zeta-Probe membrane onto the
gel surface, making contact first in the center, then allowing the
edges to gradually fold down. Carefully flood the filter surface
with NaOH. Make sure that no bubbles are present between the
gel and the Zeta-Probe membrane.
4
LIT234C.qxd 11/3/2003 11:44 AM Page 4