Bio-Rad Zeta-Probe Membranes User Manual

Zeta-Probe

®

Blotting Membranes

Instruction Manual

Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547

LIT234 Rev C

Table of Contents

Section 1 Introduction ..........................................................1

Section 2 Nucleic Acid Blotting Protocols ..............................1

2.1 Southern Blotting (DNA Capillary Transfer) .............2

2.2 Northern Blotting (RNA Capillary Transfer)..............3

2.3 Alkaline Blotting (DNA Capillary Transfer)................4

2.4 Electrophoretic Transfer .........................................5

2.5 DNA Dot Blotting ...................................................8

2.6 RNA Dot/Slot Blotting ............................................9

2.7 DNA Alkaline Fixation ...........................................10

Section 3 Probe Recommendations ....................................10

Section 4 Hybridization Protocols for DNA Probes...............11

4.1 Standard Protocol................................................13

4.2 Formamide Protocol ............................................15

4.3 Alternative Protocol..............................................16

4.4 Oligonucleotide Protocol ......................................17

Section 5 Hybridization Protocols for RNA Probes...............19

Section 6 Probe Stripping and Rehybridization ....................21

Section 7 Troubleshooting...................................................22

7.1 Nucleic Acids.......................................................22

Section 8 Appendix .............................................................26

Section 9 References ..........................................................28

Section 10 Ordering Information ............................................29

Section 1
Introduction

Zeta-Probe blotting membranes are nylon membranes which have
unique binding and handling properties that make them ideally suited
for nucleic acid, and some protein, blotting applications.

Zeta-Probe membranes possess a high tensile strength. They won't
shrink, tear, or become brittle during transfer, baking, hybridization, or
reprobing. Zeta-Probe membranes are heat-resistant, nonflammable,
and autoclavable. Zeta-Probe membranes are naturally hydrophilic
with no added wetting agents. These membranes are resistant to a
wide variety of chemicals, including 100% formamide, 2 M NaOH, 4
M HCl, acetone, most alcohols, DMSO, DMF, and chlorinated aliphat­ic hydrocarbons. The nominal porosity of Zeta-Probe membranes is

0.45 µm. When stored at 23–25°C, Zeta-Probe membranes are
stable for at least 1 year.

When handling Zeta-Probe membranes, always wear gloves or use
forceps. After blotting, do not allow wet membranes to come in con­tact with each other. Contact may result in the transfer of blotted
nucleic acids or proteins from one membrane to the other.

Stock buffers are listed in the appendix. It is suggested that you read
the entire protocol before proceeding.

Section 2
Nucleic Acid Blotting Protocols

Several nucleic acid blotting methods are presented in this section.
Capillary blotting (Sections 2.1through 2.3) is generally used with
agarose gels, and electrophoretic transfer (Section 2.4) is used with
polyacrylamide gels. Dot blotting (Sections 2.5 and 2.6) is used for

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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

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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.

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If gels contain glyoxal, remove glyoxal adducts by vacuum baking
Zeta-Probe membrane for 1 hour at 80°C after transfer. Alternatively,

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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

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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 Blotting3(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 Zeta­Probe 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 sat­urate 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.

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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
heat exchanger.

®

electrophoretic transfer system with a

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Soak one fiber pad by squeezing it while it is sub­merged in 0.5x transfer buffer. Lay the soaked pad on
the open gel holder. Soak a piece of thick filter paper
(e.g., slab gel dryer type paper cut to the size of the
fiber pad) in the transfer buffer and place it on the fiber
pad. Place the gel on the filter paper. Hold the pre­soaked Zeta-Probe membrane with both hands so that
the middle of the membrane is sagging or bowed
downward. Allow the middle of the membrane to con­tact the gel first. Gradually lower the ends of the
membrane onto the gel. This process will expel most
bubbles from between the gel and the membrane. If
there are any remaining bubbles between the gel and
membrane, remove them by sliding a test tube or
extended gloved finger across the surface.

Note: Maintaining uniform physical contact between
the gel and membrane is of critical importance in
electrophoretic transfer.

Place a presoaked piece of thick filter paper on the
membrane followed by a presoaked fiber pad. Close
the gel holder and place it in the transfer cell so that the
membrane is on the anode side of the gel (red pole).
Add more 0.5x transfer buffer, if necessary, to bring the
buffer level to 1 cm below the electrode post.

6. Transfer at 80 V for 4 hours.
Note: For comprehensive electrophoretic transfer

instructions, including protocols, technical discussion,
and troubleshooting guide, refer to the Trans-Blot cell
operating manual.

7. After transfer, separate the membrane from the gel, rinse
the membrane briefly in 1x transfer buffer, and briefly blot

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1. Prepare the stock electrophoretic transfer buffer, 20x TAE or 5x
TBE.

2. Prepare gels for transfer immediately after electrophoresis:
A. Electrophoresis Under Denaturing Conditions

If gel electrophoresis was done under denaturing conditions
(e.g., agarose/formaldehyde gels), equilibrate the gel in 0.5x
transfer buffer for 10–15 min prior to electrophoretic transfer.

B. Electrophoresis Under Nondenaturing Conditions

1. Soak the gel in 0.2 N NaOH, 0.5 M NaCl for 30 min. For
polyacrylamide gels, be sure not to exceed 30 min,
since limited gel hydrolysis may occur with subsequent
swelling during transfer.

Note: Zeta-Probe membranes will bind nondenatured
nucleic acids. Therefore, denaturing is not mandatory
before transferring. Yet, after transferring, the blotted
Zeta-Probe membrane must be treated with NaOH.
Refer to the DNA alkaline fixation procedure (Section

2.7).

2. After base treatment, neutralize the gel by washing in 5x
transfer buffer two times, 10 min each. Then wash the
gel once in 0.5x transfer buffer for 10 min.

3. While gels are being equilibrated, soak the Zeta-Probe
membrane at least 10 min in 0.5x transfer buffer.

4. Fill the electrophoretic transfer cell to half full with 0.5x
transfer buffer, and circulate 4°C coolant through the
heat exchanger. If possible, place the cell on a magnet­ic stirring plate and add a stirbar. Circulate buffer in the
cell by stirring to maintain uniform temperature during
the run.

5. Prepare the transfer assembly.

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