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Bio-Rad
Laboratories
Zeta-Probe®GT
(Genomic Tested)
Blotting Membranes
Instruction Manual
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
L
Table of Contents
Section 1 Introduction........................................1
Section 2 Nucleic Acid Blotting Protocols........2
2.1 Southern Blotting (DNA Capillary
Transfer)...............................................2
2.2 Northern Blotting (RNA Capillary
Transfer)...............................................5
2.3 Alkaline Blotting (DNA Capillary
Transfer)...............................................6
2.4 Alkaline Blotting (RNA Capillary
Transfer)...............................................8
2.5 Vacuum Blotting (RNA or DNA) ........8
2.6 Electrophoretic Transfer ....................14
2.7 DNA Dot Blotting..............................17
2.8 RNA Dot Blotting..............................18
2.9 DNA Alkaline Fixation......................21
Section 3 Probe Recommendations.................22
Section 4 Hybridization Protocols...................23
4.1 Standard Protocol...............................25
4.2 Formamide Protocol...........................28
4.3 Alternative Protocol...........................30
Section 5 Probe Stripping and
Rehybridization................................31
Section 6 Troubleshooting................................32
Section 7 Appendix...........................................36
Section 1
Introduction
Zeta-Probe GT blotting membranes are quaternary
amine derivatized nylon membranes which have unique
binding and handling properties that make them ideally suited for nucleic acid blotting applications. ZetaProbe GT membranes possess a high tensile strength.
They will not shrink, tear, or become brittle during
transfer, baking, hybridization, or reprobing. Zeta-Probe
GT membranes are heat resistant, non-flammable, and
autoclavable. Zeta-Probe GT membranes are naturally
hydrophilic with no added wetting agents. These membranes are resistant to a 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 GT
membranes is 0.45 mm. When stored at 23–25 °C, ZetaProbe GT membranes are stable for at least 1 year.
When handling Zeta-Probe GT 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 from one membrane to the other.
Stock buffers are listed in the appendix. It is suggested that you read the entire protocol you will use
before proceeding.
1
Section 2
Nucleic Acid Blotting
Protocols
Several nucleic acid blotting methods are presented in this section. Capillary or vacuum blotting (Section
2.1, 2.2, 2.3, 2.4, and 2.5) is generally used with agarose
gels, and electrophoretic blotting (Section 2.4) is used
with polyacrylamide gels. Dot blotting (Section 2.7 and
2.8) is used for nucleic acids in solution. DNA alkaline
blotting is an alternative to Southern blotting and has
shown higher resolution and greater sensitivity in many
applications (Alkaline Blotting, Section 2.3). RNA alkaline blotting reduces experimental time and is presented in Section 2.4. DNA alkaline fixation (Section 2.8)
can be used to denature and fix DNA to Zeta-Probe GT
membranes after transfer.
2.1 Southern Blotting
(DNA Capillary Transfer)
1. Depurinate the DNA by soaking the gel in 0.25 M
HCl for 10–15 minutes (be sure that the gel is floating free in all baths).
Note: Acid depurination is only recommended for
fragments > 4 kb.
1,2
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 minutes at room temperature.
3. Neutralize the gel by bathing it in 0.5 M Tris-HCl,
pH 7.4, 3 M NaCl for 30 minutes at room temperature on a moving platform. Cut five sheets of
Whatman 3MM paper and one sheet of Zeta-Probe
GT membrane to the exact size of the gel. Place
the cut sheet of Zeta-Probe GT membranes into
distilled water for 5 minutes before using.
4. Place a sponge larger than the gel in the bottom of
a deep dish or tub (two smaller sponges can be
aligned together snugly without sacrificing transfer
efficiency). Some commercial sponges have been
presoaked in detergent and should be washed well
before using. Place enough 10x SSC buffer in the
transfer dish to immerse the sponge about half way
up its side.
5. Place three sheets of the pre-cut 3MM Whatman
paper on the sponge. Flood the paper surface with
buffer and place the gel on it. Remove any bubbles
from beneath the gel by pressing them away to the
sides.
2
3
6. Spread plastic wrap over gel/3MM/sponge stack.
Cut out a window with a new razor blade, allowing
only the gel to emerge from the window . This procedure will insure capillary action only through the
gel.
7. Flood the surface of the gel with buffer and place
the pre-wetted Zeta-Probe GT membrane on it.
Carefully remove bubbles from underneath the blotting membrane, as they will block transfer. To avoid
trapping bubbles, place the Zeta-Probe GT membrane onto the gel surface by first bowing the membrane diagonally and aligning the opposite corners
with the gel corners. Then lower the membrane
onto the gel. Remove any bubbles from beneath
the membrane by pressing them away to the side.
8. Flood the blotting membrane surface with buffer.
W et the two remaining pre-cut sheets of Whatman
paper and place them on top of the blotting membrane. Align them carefully so that they completely cover the blotting membrane and do not touch the
gel directly. Remove any bubbles from beneath
each layer of filter paper.
9. Carefully place paper towels over the Whatman
paper. Stack paper towels about 15 cm high.
11. Keep an excess of buffer in the dish, but do not
cover the top of the sponge. Continue transferring
for 2–24 hours, depending on the gel concentration and fragment size.
12. After transfer, separate the membrane from the gel,
rinse the membrane briefly in 2x SSC, and air dry
the membrane. Dry the blotted Zeta-Probe GT membrane at 80 °C for 30 minutes or crosslink the DNA
to the membrane in a GS Gene Linker. The dried
membranes are stable at room temperature. The
membrane can be stored dry between two filter
papers in plastic bags at 23–25 °C.
2.2 Northern Blotting (RNA Capillary
Transfer)
Follow Southern Blotting protocol (Section 2.1),
omitting steps 1–3. If large RNA doesn’t transfer,
treat the gel with 10 mM NaOH for 10–15 minutes to
facilitate transfer to the membrane. In this case, continue on with Step 3 of Section 2.1.
10. 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.
4
5
2.3 Alkaline Blotting3(DNA Capillary
Transfer)
1. Depurinate the DNA by soaking the gel in 0.25 M
HCl for 10–15 minutes.
Note: Acid depurination is only recommended for
fragments > 4 kb.
2. Cut four sheets of Whatman 3MM paper so that
they overhang the bottom of the gel tray by 5 cm on
each end. Pre-wet Zeta-Probe GT membrane in distilled water for 5 minutes.
6. Lower the sheet of pre-wetted Zeta-Probe GT 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 gel
and membrane.
7. Cut two pieces of 3MM exactly to the gel size. Wet
a sheet of pre-cut 3MM paper in water and place it
onto the Zeta-Probe GT membrane/gel stack, then
repeat with the second sheet. Remove any bubbles
from beneath each layer of filter paper.
3. Place the four sheets of Whatman 3MM paper on an
inverted gel casting tray. Place the 3MM/tray in
the bottom of a deep dish. Then saturate the 3MM
with 0.4 M NaOH. Remove bubbles by repeatedly
rolling a test tube over the saturated 3MM. 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 onto the saturated filter
paper. Make sure that no bubbles are trapped
beneath the gel. Cover the gel with a small amount
of NaOH.
5. Place plastic wrap over the entire gel/3MM stack.
Cut a window out with a new razor blade, allowing
only the gel to emerge.
6
8. Place a stack of pre-cut paper towels on the
3MM/membrane/gel stack. Cover the paper towel
stack with a 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.
10. After transfer, separate the membrane from the gel,
rinse the membrane briefly in 2x SSC, and air dry
the membrane. Dry the blotted Zeta-Probe GT membrane at 80 °C for 30 minutes or crosslink the DNA
to the membrane in a GS Gene Linker. The dried
membranes are stable at room temperature. The
membranes can be stored dry between two filter
papers in plastic bags at 23–25 °C.
7
2.4 Alkaline Northern Blotting
(RNA Capillary Transfer)
The following protocol uses a mild alkali (NaOH)
as an RNA transfer solvent. This low NaOH concentration will cause limited hydrolysis of RNA which permits quantitative elution of large RNA molecules. This
protocol is not recommended for RNA below 5 kb. The
NaOH also removes glyoxal from RNA and insures
single stranded RNA binding.
1. Glyoxal gel—follow instructions for DNA
Alkaline Blotting (Section 2.3) except:
a. Omit step 1.
b. Use 10 mM NaOH instead of 0.4 M NaOH
as a transfer solvent in steps 3–6.
c. Transfer for 3–8 hours in step 9. Less time
is better to minimize hydrolysis.
2. Formaldehyde gel—follow instruction for DNA
Alkaline Blotting (Section 2.3) except:
a. Omit step 1.
b. Use 50 mM NaOH instead of 0.4 M
NaOH as a transfer solvent in steps 3–6.
c. Transfer for 3–8 hours in step 9. Less time
is better to minimize hydrolysis.
2. Cut a Zeta-Probe GT membrane 0.5 cm bigger than
each border of the precut window on the Window
Gasket. Cut a sheet of filter paper the same size as
the membrane.
Note: The larger the membrane/filter paper, the
easier it is to center the Window Gasket on
top of the membrane.
3. When cutting a customized window from the blank
Window Gasket, make sure the window's dimensions are at least 0.5 cm smaller than the gel's
dimensions all around, i.e. if the gel is 15 x 10 cm,
then the maximum window size should be 14 x 9 cm.
4. Fill the wells of the agarose gel with melted agarose
gel of equal concentration. Allow agarose to dry
before continuing.
5. There are two transfer procedures listed, the
Standard Transfer Procedure and the Rapid T ransfer
Procedure. The Standard Transfer Procedure is for
detecting a single copy gene in genomic DNA. On
the other hand, the Rapid Transfer Procedure is for
fast identification of DNA inserts from various
cloned vectors. For RNA transfers, follow the procedure outlined in RNA Transfer Procedure.
2.5 Vacuum Blotting (RNA or DNA)
1. Set up the Model 785 Vacuum Blotter according
to the manual.
8
9
Standard Transfer Procedure:
1. Depurinate the gel in 0.25 N HCl for 5–15 minutes
in a tray. Cover the gel with 0.25 N HCl and shake
gently.
2. Remove the 0.25 N HCl solution. Rinse the gel
twice with deionized distilled water.
3. Denature the gel in 0.5 N NaOH for 30 minutes.
Cover the gel with 0.5 N NaOH and shake gently.
4. Transfer the gel in 10x SSC for 90 minutes at 5
inches Hg. Skip to step 6.
Rapid Transfer Procedure:
1. Depurinate the gel in 0.25 N HCl for 5–15 minutes
in a tray. Cover the gel with 0.25 N HCl and shake
gently.
2. Remove the 0.25 N HCl solution. Rinse the gel
twice with deionized distilled water.
3. Immediately transfer in 0.5 N NaOH + 0.6 N NaCl
for 90 minutes at 5 inches Hg. Skip to step 6.
RNA Transfer Procedure:
1. After electrophoresis, rinse the RNA gel in deionized distilled water.
2. Soak the RNA gel in 50 mM NaOH for 5–15 minutes depending on fragment size.
3. Transfer in 10x SSC for 90 minutes at 5 inches of
Hg. Continue with Step 6.
6. Wet the pre-cut Zeta-Probe GT membrane in double distilled water by slowly lowering the membrane at a 45 degree angle to the water. Then, wet
the membrane and the filter paper in the appropriate transfer solution from the section above.
7. Make sure that the Porous Vacuum Plate lays flush
on the Vacuum Stage. Place the wetted filter paper
on the Porous Vacuum Plate. Lay the filter paper in
the area where the cut window of the Window
Gasket will be. Place the wetted membrane on top
the filter paper. Remove any air bubbles by rolling
a 10 ml glass pipet over the membrane.
8. Wet the Reservoir Seal O-ring with water.
9. Put the Window Gasket on top of the membrane/filter paper. Make sure the Window Gasket covers
the entire O-ring on the Vacuum Stage. At the same
time, make sure the membrane/filter paper is overlapping the Window Gasket. Realign as necessary.
Alternatively, place the Window Gasket on the
Porous Vacuum Plate first. Adjust the Window Gasket
to cover the entire O-ring. Then, hold one end of the
Window Gasket and slowly peel the other end back
until the window area is up off the Porous Vacuum
Plate. Place the wet membrane/filter paper on the Porous
Vacuum Plate under the window area and under the
10
11
Window Gasket. Finally, replace the partially peeled
back Window Gasket over the membrane/filter paper.
Make sure the Window Gasket covers the entire O-ring
and overlaps the membrane/filter paper on all sides.
14. With a finger , apply gently pressure on top of the gel
along the window border. This pressure helps to
form a tight vacuum seal between the gel and the
Window Gasket.
10. Gently place the gel, well side up, on top of the
Window Gasket. The gel must overlap the window.
Remove air bubbles by using a 10 ml glass pipet. As
a final check, make sure the gel edges overlap the
Window Gasket by at least 5 mm.
11. Place the Sealing Frame on top of the Vacuum
Stage. Lock the Sealing Frame onto the four latch
posts. The spring latch handle of the Sealing Frame
has a precut region in the middle. Push down on
this exposed area of the Sealing Frame with your
thumb until the latches snap onto all four latch
posts.
12. Unscrew (counterclockwise) the Vacuum Regulator
bleed valve several turns to prevent strong initial
vacuum.
13. Start the vacuum source and slowly turn the bleed
valve clockwise until the gauge reads 5 inches of
Hg. If Bio-Rad's Vacuum Pump is used, prewarm
the pump for 10 minutes before blotting. Without
prewarming, the vacuum pump will slowly increase
the pressure and then stabilize after 10 minutes.
Adjust the pressure to 5 inches of Hg.
15. Gently pour 1,000–1,500 ml of the appropriate
transfer solution (refer to step 5) into the upper
reservoir. Check to see if the gel is displaced. The
gel should be stuck to the Window Gasket. If the gel
floats, disassemble the Sealing Frame to drain the
transfer solution and repeat step 10 to 15 again.
16. Place the lid on the V acuum Blotter. Occasionally,
check the buffer level. It should be higher then the
gel. Check the vacuum pressure and adjust the to 5
inches of Hg as needed.
17. Transfer the gel for 90 minutes at 5 inches of Hg.
18. After transfer, disassemble the V acuum Blotter and
remove the Zeta-Probe GT membrane. Rinse the
membrane briefly in 2x SSC, and air dry the membrane. Dry the blotted Zeta-Probe GT membrane
at 80 °C for 30 minutes or crosslink the DNA to
the membrane in a GS Gene Linker. The dried membranes are stable at room temperature. The membranes can be stored dry between two filter papers
in plastic bags at 23–25 °C.
12
13
2.6 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.
1. Prepare the stock electrophoretic transfer buffer,
20x TAE or 5x TBE.
2. Prepare the 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 minutes
prior to electrophoretic transfer.
Soak the gel in 0.2 N NaOH, 0.5 M NaCl for
30 minutes. For polyacrylamide gels, be sure not to
exceed 30 minutes, since limited gel hydrolysis may
occur with subsequent swelling during transfer.
Note: Zeta-Probe GT membranes will bind non-dena-
tured nucleic acids. Therefore, denaturing is not
mandatory before transferring. Yet, after transferring, the blotted Zeta-Probe GT membrane
must be treated with NaOH. Refer to DNA
Alkaline Fixation (Section 2.7).
After base treatment, neutralize the gel by washing in 5x transfer buffer two times, 10 minutes each.
Then wash the gel once in 0.5x transfer buffer for
10 minutes.
3. While gels are being equilibrated, soak the ZetaProbe GT membrane at least 1 minute in 0.5x transfer buffer.
4. Fill the electrophoretic transfer cell half full of 0.5x
transfer buffer and circulate 4 °C coolant through the
heat exchanger. If possible, place the cell on a mag netic stirring plate and add a stirring bar. Circulate
buffer in the cell by stirring to maintain temperature
during the run.
b. Electrophoresis Under Non-Denaturing
Conditions
14
5. Prepare Transfer Assembly.
15
Soak one fiber pad by squeezing it while it is submerged in 0.5x transfer buffer. Lay the soaked pad on
the opened 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 lay it on the fiber
pad. Place the gel on the filter paper. Hold the presoaked membrane with both hands so that the middle of
the Zeta-Probe GT 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 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 2x SSC, and air dry
the membrane. Dry the blotted Zeta-Probe GT membrane at 80 °C for 30 minutes or crosslink the DNA
to the membrane in a GS Gene Linker. The dried
membranes are stable at room temperature. The
membrane can be stored dry between two filter
papers in plastic bags at 23–25 °C.
2.7 DNA Dot/Slot Blotting
When Zeta-Probe GT membrane is used, it is not
necessary to extract DNA from tissue samples for dot
blot analysis. Regardless of whether the sample is purified DNA (covalent circles, dsDNA, ssDNA), whole
blood, tissue, or cultured cells, it can simply be heated
in alkali then filtered directly onto Zeta-Probe GT membrane.
1. Heat the sample in a total volume of 0.5 ml with a
final concentration equal to 0.4 M NaOH, 10 mM
EDT A at 100 °C for 10 minutes.17The sample may
be purified or crude DNA (≤ 5 mg), whole soft tissue, e.g., liver (≤ 0.5 mg), whole blood (≤ 10 ml),
or cultured cells (≤ 105cells).
16
17
2. Wet a sheet of Zeta-Probe GT membrane by
immersing it in distilled water.
3. Assemble the microfiltration apparatus with a prewetted Zeta-Probe GT membrane. Make sure that
all the screws or clamps have been tightened under
vacuum to insure no cross well contamination.
4. Rinse wells with 0.5 ml TE or H2O. Apply vacuum
until wells are empty but not dry.
5. Apply 0.5 ml DNA samples into appropriate wells
without vacuum. Apply vacuum until the wells are
just dry.
6. Rinse all wells by placing 0.5 ml of 0.4 M NaOH in
each, then apply vacuum until all wells are quite dry.
7. Without disconnecting the vacuum, disassemble
the apparatus and remove the membrane. Then rinse
the membrane briefly in 2x SSC and allow it to air
dry. Dry the blotted Zeta-Probe GT membrane at
80 °C for 30 minutes or crosslink the DNA to the
membrane in a GS Gene Linker. The dried membranes are stable at room temperature. The membranes can be stored dry between two filter papers
in plastic bags at 23–25 °C.
2.8 RNA Dot/Slot Blotting
Both native and denatured RNA are retained quantitatively by Zeta-Probe GT membrane. However to
insure optimal hybridization, RNA samples must be
totally denatured before fixing onto the Zeta-Probe GT
membrane. T wo protocols are presented for denaturing
RNA samples. The first protocol uses a mild alkali for
denaturing and fixing the RNA sample. The second
protocol uses glyoxal for denaturation.
Alkaline RNA Denaturation and Fixation
1. Immediately before use, dissolve the RNA samples in 0.5 ml total volume of ice-cold 10 mM
NaOH, 1 mM EDTA final concentrations.
2. Assemble microfiltration apparatus with a sheet of
Zeta-Probe GT membrane pre-wetted in distilled
water. Make sure that all the screws or clamps have
been tightened under vacuum to insure no cross
well contamination.
3. Rinse wells with water or TE, apply vacuum until
wells are just dry.
4. Apply 0.5 ml samples to wells without vacuum.
5. Apply vacuum until wells are just dry, then release
the vacuum.
6. Add 0.5 ml of cold 10 mM NaOH, 1 mM EDTA to
each well and then apply vacuum until the wells
are dry.
7. With vacuum on, disassemble and remove the membrane.
18
19
8. Immediately rinse the membrane in 2x SSC, 0.1%
SDS and blot lightly. Dry the blotted Zeta-Probe
GT membrane at 80 °C for 30 minutes or crosslink
the DNA to the membrane in a GS Gene Linker.
The dried membranes are stable at room temperature. The membranes can be stored dry between
two filter papers in plastic bags at 23–25 °C.
7. Apply vacuum until wells are just dry, then release
vacuum.
8. Rinse all wells with 0.5 ml TE, applying vacuum
until completely dry.
9. Disconnect the vacuum. Remove blotted Zeta-Probe
GT membrane.
Glyoxal RNA Denaturation and Fixation
1. Add RNA sample to the following final
concentrations:
50% dimethyl sulfoxide (DMSO)
10 mM sodium phosphate, pH 7.2
1 M glyoxal
2. Incubate sample for 1 hour at 50 °C. Then cool the
RNA sample on ice.
3. Wet a sheet of Zeta-Probe GT membrane by
immersing it in distilled water.
4. Assemble the microfiltration apparatus with prewetted Zeta-Probe GT membrane. Make sure that
all the screws or clamps have been tightened under
vacuum to insure no cross well contamination.
5. Rinse wells with TE or water, apply vacuum until
just dry.
6. Apply 0.5 ml RNA samples to appropriate wells
without vacuum.
20
10. Rinse the membrane briefly in 2x SSC, and air dry
the membrane. Dry the blotted Zeta-Probe GT membrane at 80 °C for 30 minutes or crosslink the DNA
to the membrane in a GS Gene Linker. The dried
membranes are stable at room temperature. The
membranes can be stored dry between two filter
papers in plastic bags at 23–25 °C.
Alternatively, place the membrane in a vacuum oven
at 80 °C for 1 hour (omit the above step 9).
2.9 DNA Alkaline Fixation
After transfer, place the Zeta-Probe GT membrane
(DNA surface uppermost) on a pad of filter paper saturated
with 0.4 M NaOH for 10 minutes. Rinse in 2x SSC and
air dry. The dried membranes are stable at room temperature. Crosslinking is not necessary when nucleic acids are
applied under alkaline conditions.
21
Section 3
Probe Recommendations
The specific activity, concentration, size range, and
purity of the probe all have an important effect on signal-to-noise ratio during hybridization. For hybridization on Zeta-Probe GT blotting membranes the following
is recommended:
Probe specific activity 108cpm/µg probe
Probe concentration in
the hybridization mixture 106cpm/ml (10–15 ng/ml)
Probe length 200–1,000 bp
Probe clean-up is essential to minimize background.
Unincorporated nucleotides present after probe preparation contribute to hybridization background. The most
effective cleanup method is desalting by column separation. This can be done in a column 1 to 5 ml bead
volume using Bio-Gel®P-30 gel (catalog number
150-1340) or with Bio-Spin®30 columns (catalog number 732-6004).
After clean-up, denature double stranded probes by
increasing temperature to 95–100 °C for 5 minutes.
Then cool rapidly in ice. Use the probe as soon as possible after preparation.
Probe length is an important parameter to control.
DNA probes prepared by random priming tend to be
small. Small probes can cause lane specific background
during low stringency hybridization. DNA probes prepared by nick translation are generally long. Probe fragments longer than 1 kb decrease hybridization
specifically.
Template purity is essential during probe synthesis, especially probes made by random primer extension. Small amounts of contaminating DNA templates
can cause lane background or extra bands due to the
high specific activity of random priming.
Optimal probe specific activity and concentration
can vary according to available hybridization sites and
exposure time.
22
Section 4
Hybridization Protocols
There are several hybridization protocols that will
give high quality results. The key to successful nucleic acid blotting is proper blocking of the Zeta-Probe
GT membrane. The most important blocking reagent
in the hybridization solution is sodium dodecyl sulfate
(SDS). SDS is the most effective blocking reagent when
used at concentration ≥1% (w/v). The Standard Protocol
(Section 4.1) uses 7% (w/v) SDS, which has been shown
to give extremely low background and high signals.
The protocol described in Section 4.2 includes formamide, which allows hybridization to be performed at
a lower temperature. Oligonucleotide probes can be
used with the Standard Protocol (Section 4.1). Just cal-
23
culate the proper temperature with the following formulas. The Alternative Protocol (Section 4.3) should
be used only when extreme sensitivity is needed.
The final volume of hybridization solution is also
important in reducing background. During hybridization,
use 70–150 µl solution/cm2Zeta-Probe GT membrane
and use at least 2 ml solution /cm2Zeta-Probe GT membrane for washes.
One of the most significant advantages offered by
Zeta-Probe GT membrane over conventional membranes is that target nucleic acids of all sizes can be
fixed irreversibly. The stringency of hybridization can
therefore be optimized for detection of specific target
sequences. There is no need to use high ionic strength
and low temperature to minimize the loss of nucleic
acids from the membrane during hybridization or washing procedures.
Tm(DNA/DNA) = 81.5 + 16.6 x log[Na+] - 0.65 x
(% formamide) + 41 x (G+C).
11
Tm(RNA/RNA) = 79.8 + 18.5 x log[Na+] - 0.35 x
(% formamide) + 58.4 x (G+C) + 11.8 x (G+C)2 - 820/L.
12
Tm(DNA/RNA) = approximate mean of Tm(DNA/DNA
and Tm(RNA/RNA).
The Tmis decreased approximately 1.5 °C for every
1% decrease in homology.
10,11
The Tmis decreased as the fragment length of the probe
decreases: the appropriate correction factor is approximately -500/(#bp in probe fragment) °C.
10, 11
The rate of hybridization increases as the salt concentration increases.
10
The rate of hybridization decreases as the formamide
concentration increases.
10, 13
Hybridization should be conducted at 20–25 °C
below the melting temperature (Tm) of the probe duplex,
to insure optimal rates of specific hybridization while
minimizing interaction with partially homologous
sequences.10The stringency of post-hybridization washes is less critical, but a good rule of thumb is to conduct
the most stringent wash at 10–15 °C below Tm.11The
protocols described below are suitable for probes having a (G + C) content representative of the mammalian
genome, i.e., 0.42. If desired, conditions can be varied
in accordance with the following empirical formula:
24
The hybridization temperature (TH) appropriate to synthetic oligomeric DNA probes below 21 bases can be
approximated by the following:
TH= 2 x (no. of A-T bp) + 4 x (no. of G-C bp) - 5.
14
4.1 Standard Protocol
This hybridization protocol can be used with
DNA/DNA, DNA/RNA, and RNA/RNA hybrids. The
Standard Protocol can also be used with oligonucleotide
probes by adjusting the THfrom the previous section.
25
Prehybridization:
Washes:
1. Seal blotted Zeta-Probe GT membrane inside a heat
sealable plastic bag.
2. Cut one corner of the plastic bag and pipet prehybridization solution in:
0.25 M sodium phosphate, pH 7.2
7% SDS
3. Seal the bag and incubate briefly at 65 °C for
5–30 minutes. The goal is simply to coat the membrane evenly and completely with this solution.
Hybridization:
1. Cut one corner of the plastic bag, remove the prehybridization solution, and replace it with same
buffer.
2. Add denatured probe and remove all air bubbles
before resealing the bag. Hybridize for 4–24 hours
at 65 °C with agitation.
3. Carefully remove the hybridization solution by cutting one corner. Remove the hybridized Zeta-Probe
GT membrane from the plastic bag.
Note: At no stage before washing should the mem-
branes be permitted to dry.
1. W ash membrane at 65 °C, 2 times, for 30–60 minutes each, in the following:
20 mM sodium phosphate, pH 7.2
5% SDS
2. Wash membrane at 65 °C, 2 times, for 30-60 minutes each, in the following:
20 mM sodium phosphate, pH 7.2
1% SDS
3. After washing, the blotted membranes are ready
for autoradiography. If no further cycles of
hybridization are to be done on the membrane, then
the membrane can be dried. When reprobing, do
not allow the membrane to dry between hybridizations. Expose moist membranes between plastic
wrap or enclosed in a sealable plastic bag. Do not
allow a wet membrane to come in contact with the
film, because wet Zeta-Probe GT membrane will
stick to the film.
26
27
4.2 Formamide Protocol
Prehybridization:
1. Seal blotted Zeta-Probe GT membrane inside a heat
sealable plastic bag. Combine these reagents in the
order given. For 100 ml:
50 ml formamide
25 ml 0.5 M sodium phosphate, pH 7.2
15 ml H2O
1.46 g NaCl
7.0 g SDS
2. Add probe, then seal the open corner (taking care
to exclude all air bubbles). Mix the contents of the
bag thoroughly. Incubate at 43 °C for 4–24 hours
with agitation.
Note: At no stage before washing should the mem-
branes be permitted to dry.
Washes:
1. At the completion of hybridization, remove the
membranes from their hybridization bags into
2x SSC. Rinse briefly , then wash them successively by vigorous agitation at room temperature for
15 minutes in each of the following solutions;
Bring to 100 ml with H2O. If precipitation occurs,
heat the solution as necessary.
2. Cut one corner of the plastic bag and pipet in prehybridization solution, then reseal the plastic bag.
3. Incubate at 43 °C for 5 minutes. The goal is simply
to coat the membrane evenly and completely with
this solution.
Hybridization:
1. Cut one corner of the bag, remove the prehybridization solution, and replace it with the same
buffer.
28
2x SSC/0.1% SDS
0.5x SSC/0.1% SDS
0.1x SSC/0.1% SDS
Note: For single copy detection or high stringency , con-
duct the last wash at 65 °C.
29
2. After washing, the blotted membranes are ready
for autoradiography. If no further cycles of
hybridization are to be done on the membrane, the
membrane can be dried. When reprobing, do not
allow the membrane to dry between hybridization.
Expose moist membranes between plastic wrap or
enclosed in a sealable plastic bag. Do not allow a
wet membrane to come in contact with the film,
because wet Zeta-Probe GT membrane will stick
to the film.
4.3 Alternative Protocol
When extreme hybridization sensitivity is needed,
this accelerator will help increase the target signal by acting as volume excluder. A hybridization accelerator
will also decrease the hybridization time needed. In
some applications, an accelerator can reduce the
hybridization time from overnight to 4 hours. It is suggested that you first work with the standard hybridization protocol (Section 4.1) and determine if your
experiments require hybridization accelerators before
using the following protocols.
Polyethylene glycol (PEG)
tions for standard hybridization (Section 4.1) or
formamide hybridization (Section 4.2) except add 10%
(w/v) PEG 8,000 mw, into the hybridization solution in
step 1. For example, 1 g of PEG 8,000 plus hybridization
solution to 10 mls. Conduct post hybridization washes the
same as in Section 4.1 or 4.2, without PEG.
15,16
—follow the instruc-
Section 5
Probe Stripping And
Rehybridization
If reprobing is desired, do not allow the Zeta-Probe
GT membrane to dry between hybridizations.
The Zeta-Probe GT membrane should be stripped
as soon as possible after autoradiography.
W ash the membrane twice for 20 minutes each in a
large volume of 0.1x SSC/0.5% SDS at 95 °C.
Check membrane by overnight exposure.
DNA Blots Only
Alternatively, soak a pad of filter papers in 0.4 N
NaOH. Place the membrane DNA side up onto the
soaked filter papers for 5 minutes. Rinse in 2x SSC.
Repeat.
Check membrane by overnight exposure.
2
30
31
Section 6
Zeta-Probe GT Membrane
Troubleshooting Guide
Problem Solution
1. Fragments greater
than ~1,000 bp can
not be electrophoretically transferred from
polyacrylamide gels
after base denaturation, even at increased
volts/hours.
We have observed that fragments >~1,000 bp can
become trapped in polyacrylamide gels if they are base
denatured and neutralized
after electrophoresis, whereas those not denatured prior
to and during electrophoresis
will transfer completely up to
at least 2,000 bp. Solve this
problem in any of three ways:
1. Omit pretreatment, and
transfer dsDNA. Alkaline fix
(Section 2.8) post blotting.
2. Run gel electrophoresis
under denaturing conditions and omit base denaturation step and neutralization step prior to transfer.
3. Omit base denaturation
step, and denature gel
instead with 1 M glyoxal, in
25 mM sodium phosphate,
pH 6.5, 50% DMSO for
1 hour at 50 °C. Then
transfer directly.
Problem Solution
2. Very large fragments
cannot be electrophoretically eluted from
agarose gels.
3. High background
observed throughout
membrane on autoradiograph.
1. Solutions 1 and 2 to problem 1 can also be applied
to agarose gels.
2. Depurinate prior to transfer
by soaking the gel in 0.25 M
HCl for 20 minutes.
The major contributors to
background are unincorporated label, small radioactive
decay products, and small
probe fragments resulting
from nick-translation or random priming. Proper blocking
of the membrane is very
important, use ≥1% SDS. We
recommend 7% SDS. Refer to
hybridization protocols.
1. Use a desalting gel column
to remove unincorporated
label, or the Bio-Spin 30
columns.
2. Use the probe as soon as
possible after preparation,
since decay results in fragmentation.
3. Use a different heterolo-
gous nucleic acid in the prehybridization and
hybridization mixtures.
Sonicate it thoroughly and
denature it before use.
32
33
Problem Solution
4. Localized high background observed on
autoradiograph.
5. Lane background or
extra bands.
1. Make sure the membrane
is free floating within the
plastic bag during
hybridization.
Membrane/bag contact
during hybridization can
cause background. Add
more hybridization solution.
2. Make sure not to pinch the
membrane when sealing
plastic bag prior to
hybridization.
3. Be sure no bubbles exist in
hybridization bag.
1. Contaminated template.
Make sure the probe is
synthesized with the pure
template of choice.
Sequence the probe; if the
probe contains a repeat
sequence add carrier
DNA/RNA.
Problem Solution
6. Low autoradiograph
signal.
7. No autoradiograph
signal.
This problem may occur
when total genomic DNA is
probed for single copy or
low copy number genes.
1. Incorporate 10% PEG in
the hybridization mixture.
This polymer effectively
reduces the solvent volume, thereby increasing
the concentration of the
solutes and enhancing
hybridization.
2. Increase exposure time to
increase signal-to-noise ratio.
3. Increase sample load on
the gel.
4. If low signal is accompanied by low background,
probe concentration can
be increased 2 to 4-fold.
1. After transfer, stain the gel
to check that transfer was
complete. If not, increase
transfer time and/or voltage
of transfer, or see solution
to problem 1.
2. Be sure probe is denatured
by boiling or heating to
65 °C for 5 minutes in 50%
formamide prior to
hybridization.
34
35
Section 7
Appendix
20x TAE MW g/l
800 mM Tris Base 121.1 96.9
400 mM Na acetate 82.04 32.8
20 mM EDTA 372.2 7.45
pH to 7.4 with glacial acetic acid
5x TBE MW g/l
0.5 M boric acid 61.8 30.9
0.5 M Tris Base 121.1 60.5
10 mM EDTA 372.2 3.73
20x SSC MW g/l
3 M NaCl 58.44 175
0.3 M trisodium citrate 294.1 88.2
20x SSPE MW g/l
3.6 M NaCl 58.44 210
0.2 M Na2HPO47 H2O 268.07 53.6
0.02 M EDTA 372.2 7.44
20% SDS MW g/l
20% Sodium dodecyl 288.38 200
sulfate
Heat to 65 °C to get into solution
0.5 M sodium
phosphate, pH 7.2 FW g/l
Stock A:
0.5 M NaH2PO4•H2O 138.0 69
Stock B:
0.5 M Na2HPO4•7H2O 268.1 134
Combine 316 ml of Stock A with 684 ml Stock B to
make 1 liter of 0.5 M sodium phosphate pH 7.2.
Refer to reference 18.
50% Formamide g/100 ml
50% formamide 50
Store at 4 °C. Immediately before use, deionize the
required volume by stirring gently for 1 hour with 1 g
mixed-bed ion exchange resin (AG®501-X8 (D) resin,
catalog number 142-6425)/ 10 ml of formamide. Filter
through coarse filter paper.
TE
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
6 M Glyoxal (deionized)
Deionize 6 M glyoxal by pouring over a small
mixed-bed resin column (AG 501-X8 resin, catalog
number 142-6425). Store at -20 °C in small aliquots.
If the aliquot is exposed to the air, it cannot be reused.
36
37
Ordering information
Catalog
Number Product Description
162-0190 Zeta-Probe GT Membrane, 9 x 12 cm,
15 sheets
162-0191 Zeta-Probe GT Membrane, 10 x 15 cm,
15 sheets
162-0192 Zeta-Probe GT Membrane, 15 x 15 cm,
15 sheets
162-0193 Zeta-Probe GT Membrane, 15 x 20 cm,
15 sheets
162-0194 Zeta-Probe GT Membrane, 20 x 20 cm,
15 sheets
162-0195 Zeta-Probe GT Membrane, 20 x 25 cm,
15 sheets
162-0196 Zeta-Probe GT Membrane, 30 cm x 3.3 m
roll
162-0197 Zeta-Probe GT Membrane, 20 cm x 3.3 m
roll
165-5000 Model 785 Vacuum Blotter with Regulator
170-3910 Trans-Blot Electrophoretic Transfer Cell
165-5052 PowerPac 200 Power Supply, 100/120 V
170-6545 Bio-Dot
170-6542 Bio-Dot SF Microfiltration Apparatus
®
Microfiltration Apparatus
38
Catalog
Number Product Description
142-6425 AG 501-X8 (D) Mixed Bed Ion
Exchange Resin
150-1340 Bio-Gel P-30 Fine Mesh Filtration Gel
732-6004 Bio-Spin 30 Columns
References
1. Southern, E. M., J. Mol. Biol., 98, 503 (1975).
2. Gatti, R. A., Concannon, P. and Salser, W., BioTechniques, May/June
(1984).
3. Reed, K. C. and Mann, D. A., Nucleic Acids Res., 13, 7207-7221 (1985).
4. Church, G. M. and Gilbert, W., Proc. Natl. Acad. Sci. USA, 81, 1991
(1984).
5. Thomas, P. S., Proc. Natl. Acad. Sci. USA, 77, 5201 (1980).
6. Angelini, G., Proc. Natl. Acad. Sci. USA, 83, 4489 (1986).
7. Plapp, F. V., Rachel, J. M. and Sinor, L. T., The Lancet, June 28 (1986).
8. Denhardt, D., Biochem. Biophys. Res. Commun., 23, 641 (1966).
9. W ahl, G. M., Stern, M. and Stark, G. R., Proc. Natl. Acad. Sci. USA, 76,
3683 (1979).
10. Britten, R. J., Graham, D. E. and Neufeld, B. R., Methods Enzymology,
29, 363-418 (1974).
11. Beltz, G. A., Jacobs, K. A., Eickbush, T. H., Cherbas, P. T. and Kafatos,
F. C., Methods Enzymology, 100, 266 (1983).
12. Bodkin, D. K. and Knudson, D. L., Virology, 143, 55 (1985).
13. Casy, J. and Davidson, N., Nucl. Acid Res., 4, 1539 (1977).
14. Berent, S. L., Mahmoudi, M., Torczynski, R. M., Bragg, P. W . and Bollon,
A. P., BioTechniques, 3, 208 (1985).
15. Amasino, R. M., Anal. Biochem., 152, 304-307 (1985).
16. Budowle, B., Applied & Theoretical Electr ophor esis , 1, 181- 187 (1990).
17. Reed, K. C. and Matthaei, K. I., Nucleic Acids Res., 18, 10 3093 (1990).
18. Sambrook, J., Fritsch, E. F., and Maniatis, T., In Molecular Cloning, A laboratory Manual. Cold Spring Harbor Laboratory Press (1989).
39
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