Bio-Rad Zeta-Probe Membranes - Manual (Page 4)

diagonally and aligning the opposite corners with the gel corners.
Then lower the Zeta-Probe membrane onto the gel.
6. Cut two pieces of 3MM paper to the size of the gel. Place both
sheets on top of gel. Wet paper with small amount of transfer
buffer. Roll with the pipet to remove air bubbles.
7. Flood the surface of the gel with buffer. Carefully place paper
towels over the Whatman paper. Stack the paper towels about
15 cm high.
8. Cover the paper towel stack with a glass or plastic plate. Keep
the pressure on the paper towel stack at a minimum. Excessive
weight will compress the gel, retarding capillary transfer.
9. Keep an excess of buffer in the dish, but do not cover the top of
the sponge. Continue transferring for 2–24 hr, depending on the
gel concentration and fragment size.
10. After transfer, separate the membrane from the gel, rinse the
membrane briefly in 2x SSC, and briefly blot the membrane with
filter paper. The DNA can then be fixed onto the Zeta-Probe
membrane by baking it at 80°C for 30 min in a vacuum oven.
Alternatively, the DNA can be UV-crosslinked to the membrane
using 5,000 µJ/cm
2 radiation. Higher levels, although they increase
the absolute retention of the nucleic acid on the membrane, can
lead to a reduction in signal intensity. The membranes can be
stored dry between two pieces of filter paper in plastic bags at
23–25°C.
2.2 Northern Blotting (RNA Capillary Transfer)
Follow the Southern blotting protocol (Section 2.1), omitting steps
1–3. No pretreatment of RNA gels is necessary.
5
If gels contain glyoxal, remove glyoxal adducts by vacuum baking
Zeta-Probe membrane for 1 hour at 80°C after transfer. Alternatively,
3
nucleic acids in solution. DNA alkaline blotting (Section 2.3) is an
alternative to Southern blotting. DNA alkaline blotting results in higher
resolution and greater sensitivity in many applications. DNA alkaline
fixation (Section 2.7) can be used to denature and covalently fix DNA
to Zeta-Probe membranes after transfer.
2.1 Southern Blotting 1,2 (DNA Capillary Transfer)
1. Depurinate the DNA by soaking the gel in 0.25 M HCl for
10–15 min (be sure that the gel is floating free in all baths).
Note: Acid depurination is only recommended for fragments >4 kb.
2. Denature the DNA by placing the gel in a bath of 0.5 N NaOH, 1
M NaCl. Place the container on a moving platform for 30 min at
room temperature.
3. Neutralize the gel by bathing it in 1.5 M Tris-HCl, pH 7.4,
1.5 M NaCl for 30 min at room temperature on a moving
platform. Prepare a Whatman 3MM paper wick. Hang two
sheets, prewetted with 10x SSC hung over the sides and into the
bottom of the capillary transfer apparatus containing 800 ml 10x
SSC. On top of the wick, place two additional sheets of 3MM
paper cut to the size of the gel prewetted with 10x SSC.
4. Invert the gel and place it on the wick. Roll a 10 ml plastic pipet,
over the gel to remove any bubbles. Trim off the wells from the
gel using a spatula.
5. Place membranes labeled side against the gel above the lanes to
be transferred. Trim edges of the gel as required with a spatula,
or cover exposed areas with Parafilm. Roll with the pipet to
remove any air bubbles. It is important to remove air bubbles
from underneath the blotting membrane as they will block
transfer. To avoid trapping bubbles, place the Zeta-Probe
membrane onto the gel surface by first bowing the membrane
2
LIT234C.qxd 11/3/2003 11:44 AM Page 2