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Dr. Showalter
SOUTHERN BLOT LAB: BACKGROUND, OVERVIEW & PROTOCOL
Background
In 1975, E. M. Southern developed a technique to transfer DNA directly from an agarose gel
to a nitrocellulose or nylon membrane. The technique relies on capillary action. The DNA, once
bound on the membrane, can then be used for hybridization analysis to determine the presence
and location of a particular DNA sequence. In the past decade, Southern's original paper on the
technique was the most frequently cited paper in research.
Restriction digestion, electrophoresis and staining allow us to cut DNA molecules into
reproducible fragments and to look at the sizes of those fragments. However, it does not give
much information about the DNA base sequence within the fragments. Often it is important to
know whether a specific DNA base sequence is present in a sample and where it is located with
respect to restriction sites. The technique most commonly used to address these issues is
hybridization analysis.
Hybridization analysis involves separating (denaturing) the strands of the DNA molecules to
be analyzed and mixing those strands with a labeled, single-stranded DNA or RNA probe. If the
probe's sequence is complementary to a DNA molecule, then hybridization (or annealing) can
occur under the appropriate conditions. The sample can be rinsed to remove excess probe (and
non-specific hybridization) and tested for the presence of hybridized probe. Hybridized probe
indicates the presence of the DNA sequence of interest.
One Southern blot hybridization application is the screening of DNA libraries. For example,
a genomic DNA library is a collection of clones that, taken together, represent the entire genome
of an organism. To find a plasmid containing a specific DNA sequence (or gene of interest), the
entire library is transformed into a bacterial host. The transformants are plated onto agar to
produce individual colonies, each representing one fragment of the organism's DNA, "lifted"
onto a membrane, and the DNA denatured and probed with the sequence of interest. If colonies
are identified, they can be transferred to separate cultures, and plasmid DNA prepared for further
analysis and use.
Another major application of Southern blot hybridization analysis is the testing of the
products of a restriction digest to determine which fragments, if any, contain a certain
DNA sequence, or to determine the size of a fragment containing that sequence. The
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procedure most commonly used for this is called "Southern gel blotting" and is followed
by hybridization analysis.
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Southern Blot Lab: Overview
Day
Gel hybridization
March 29
Genomic DNA isolation
(Monday)
PCR amplification (TA)
March 31
Electrophoresis & transfer
(Wednesday)
Colony Hybridization
Genomic DNA isolation
PCR amplification (TA)
Ligation and transformation
April 1
(Thursday)
Membrane cross linking and
membrane stored at 4°C
Plates stored at 4ºC
April 5
(Monday)
-
Colony lifting
April 7
(Wednesday)
Probe labeling and
overnight incubation at
37°C
Probe labeling and
overnight incubation at
37°C
April 8
(Thursday)
April 11
(Sunday)
April 12
(Monday)
Probe stored at -20ºC (TA)
Probe stored at -20ºC (TA)
Prehybridization,
hybridization and overnight
incubation at hybridization
temperature
Washes and detection
Prehybridization,
hybridization and overnight
incubation at hybridization
temperature
Washes and detection
Materials needed:
0.2 N HCl
DIG High Prime labeling and detection kit
0.5 M NaOH/1.5 M NaCl
20X, 2X SSC
1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 0.2M EDTA (pH 8.0)
Nitrocellulose membranes
Paper towels
Whatman 3MM filter paper
Glass plate
Sponge
Pyrex dish
Weight
Tupperware dish/or sealable bag
Shaking incubator or water bath, temperature controlled
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Southern Blot Lab: Detailed Protocol
Day 1
A. Genomic DNA extraction and PCR amplification
Genomic DNA was extracted using the CTAB method and the sequence of interest was
amplified using PCR.
PCR set up:
2 l Genomic DNA (25-100ng/l)
2.0 l 10X buffer
0.25 l dNTPs
0.25 l Taq DNA polymerase
1 l Forward primer
1 l Reverse primer
Add water to 20l total volume
Genomic DNA and PCR product along with the marker was run on a mini-gel for 1.5- 2h.
If you want to hybridize restriction digested genomic DNA, you need:
5 g genomic DNA
2 l 10X buffer
1 l enzyme (10 U/l)
Add water to 20 l total volume
Digest overnight at 37°C and run on a mini-gel for 1.5-2 h.
B. Gel
i.Place gel, in 0.2 N HCl for 10 min, then rinse with water
ii. Place in 0.5 M NaOH/1.5 M NaCl (Denaturation solution). Incubate twice for 15
min.
iii. Place gel in 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 (Neutralization solution). Incubate
for 30 min and rinse with water
iv. Assemble overnight blot (semi-dry blot).
v. Take apart the blot the next day and UV cross-link (or bake for 30 min at 80oC) to
bind the DNA to the membrane.
C. Setting up the gel for transfer to nitrocellulose (i.e., blotting)
(A) Sponge method transfer set up:
Place a sponge, slightly larger than the gel, in a glass tray and fill with enough 20X SSC
so that the soaked sponge is about half submerged. When the sponge is fully saturated,
layer 3 pieces of Whatman 3MM paper. Wet the filter paper thoroughly and remove all
air bubbles. Drain the gel and lay on top of the filter paper being careful to avoid air
bubbles. (Clip off one corner of your nitrocellulose and record which corner you
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removed. This will help you orient your blot to the picture of your gel.) Lay the
nitrocellulose exactly on top of the gel. Be sure the nitrocellulose does not hang over the
gel (it should fit exactly on top) and remove any trapped air bubbles. Now add, in order, 3
more pieces of Whatman filter paper, 2 inches of paper towels, a glass plate and top it off
with a small weight. Cover the edges of the tray with plastic wrap. Allow the transfer to
proceed overnight.
D. Preparation of the target
a. Colony lift
i. Carefully lay the membrane onto the pre-cooled agar plate. Beginning
at one edge of the plate lay the membrane down smoothly, avoiding
bubbles.
ii. Mark the membrane and plate with a distinctive pattern
iii. Remove the membrane from the plate after 1 min. A second lift may be
performed if necessary. Seal the master plate with parafilm and store at
4oC until needed.
iv. Place the membrane, colony-side up, onto filter paper saturated with 0.5
M NaOH/1.5 M NaCl (Denaturation solution). Incubate 5 min.
v. Briefly blot the membrane on dry filter paper.
vi. Place the membrane, colony-side up, onto filter paper saturated with 1.5
M NaCl/0.5 M Tris-HCl, pH 7.4 (Neutralization solution). Incubate for
5 min.
vii. Briefly blot on dry filter paper.
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viii. Place the membrane, colony-side up, onto filter paper saturated with 2X
SSC for 5 min.
ix. UV cross link DNA (or bake for 30 min at 80oC) to bind the DNA to
the membrane.
E. Make the probe.
a. Add 1g of the template DNA and sterile, double distilled water to a final
volume of 24 l.
b. Denature the DNA by heating in a boiling water bath for 10 min and quickly
chilling in an ice/water bath.
c. Mix DIG-High prime (vial 1) thoroughly and add 6l to the denatured DNA,
mix and centrifuge briefly
d. Incubate 1 h or overnight at 37oC
e. Stop the reaction by adding 2 l 0.2M EDTA (pH 8.0) or heating to 65oC for
10 min
Day 2
A. Hybridization
Hybridization analysis is a little like fishing (for DNA). If you know a little about the
DNA sequence you're interested in, locating it shouldn't be too difficult. Using that
sequence, you can design a probe (the "hook") and the nitrocellulose-bound DNA
provides the pond. The rest is just methodology and it is the same for a gel or colony lift.
Prehybridization and hybridization of the membrane
i. Preheat an appropriate volume (10ml/100 cm2 filter) of DIG Easy Hyb to the
appropriate hybridization temperature (to be announced).
ii. Place the nitrocellulose membrane in a sealable bag or plastic container
(Tupperware). Add enough prehybridization solution (i) to fully cover the
membrane and prehybridize 30 min, shaking gently, at the hybridization
temperature.
iii. Denature DIG-labeled probe by boiling for 5 min and rapidly cooling in an
ice/water bath.
iv. Add 15 l denatured DIG-labeled probe (iii) to the preheated DIG Easy Hyb (i)
and mix well (but gently to avoid bubbles that increase background).
v. Pour off the prehybridization solution (ii) and add probe/hybridization mixture
(v) to the membrane
vi. Incubate 4 h to overnight at the hybridization temperature with gentle agitation.
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Day 3
Washes and detection
A. Stringency washes
Rinse the filter as follows:
i. 2 X 5 min in 2X SSC, 0.1% SDS at RT. with constant agitation
ii. 2 X 15 min in 0.5X SSC, 0.1% SDS (pre-warmed to wash temperature) at 65oC
B. Detection
i. Rinse the membrane briefly (1-5 min) in Washing Buffer
ii. Incubate for 30 min in 100 ml Blocking solution.
iii. Incubate for 30 min in 20 ml Antibody solution
iv. Wash 2 X 15 min in 100 ml Washing buffer
v. Equilibrate 2-5 min in 20 ml Detection buffer.
vi. Incubate the membrane in 10 ml freshly prepared color substrate solution in the
dark. Do not shake during color development. Monitor the color development
(may take up to 16 h).
vii. Stop the reaction, when the desired spot or band intensities are achieved, by
washing the membrane for 5 min with 50 ml sterile double distilled water or TE
buffer.
viii. Results can be documented by photocopying the wet membrane or by photography.
Important Notes:
Genomic DNA was extracted from Arabidopsis mutant (Feldmann line) with a TDNA insertion (Lab 1, Dr. Wyatt lab). The T-DNA insert in the Feldmann lines is a
16,000bp known sequence flanked by left and right border repeat sequences. T-DNA
inserts into the genome of the plant causing insertional mutagenesis. The characterization
of the mutant genes depends on designing primers based on the known sequence on either
the right border or left border of the T-DNA. To make sure that an intact right border
sequence is present in the genome for the characterization studies, a 1000bp region from
the right border of the T-DNA was amplified using PCR. This is the PCR product that
was cloned into the TOPO vector (Lab 2, Dr. Wyatt’s lab). As a confirmatory step, the
agarose gel with the genomic DNA and the PCR product along with the marker after
electrophoresis will be transferred onto the nitrocellulose membrane and a Southern
hybridization will be done. The PCR product cloned into the TOPO vector will be used as
a probe for the experiment. A signal in the genomic DNA lane confirms the presence of
the right border region of the T-DNA. PCR product was run as control for the
experiment.
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Dr. Showalter
For the colony lift, the transformed E. coli cells from Dr. Wyatt’s lab (Lab 2) will be
used. The bacteria were plated on selective media that contain X-Gal for blue/white
screening. The cells contain the TOPO vector into which a part of the right border TDNA was cloned. The same probe will be used for the colony lift and the conventional
blot. The students have to identify clones that contain the insert versus cells that carry the
re- ligated vector.
The protocol for this lab was adapted from Roche DIG High Prime DNA labeling
and Detection Starter Kit 1 (Cat. No. 1 745 832 910).
Conclusions:
Did the probe recognize the genomic DNA (or colonies) from the gel? If so, what
does that mean? If not, why not?
Lab Report (100 points, due at the beginning of lab on April 19, 2010):
Your lab report should include the following sections and describe everything that
was done in the style of a PNAS paper. Each student must prepare and submit their own
lab report.
 Abstract
 Introduction (on the method and objectives)
 Results
 Discussion
 Materials and methods (summarized protocol)
 Acknowledgements
 References
 Answers to the following Questions:
1. Who invented Northern blotting and Western blotting?
2. What is a Southwestern blot?
3. Explain another way to label a DNA probe for Southern hybridization analysis?
4. What is Southern dot blot analysis and what is it used for?
5. What two hybridization parameters or conditions are typically altered for maximum
hybridization?
6. What is a zoo blot and a garden blot?
7. What kinds of nucleic acid bind to nitrocellulose? What kinds of nucleic acid do not
bind?
8. Why do you soak the gel and not the colony lift in 0.2N HCl?
9. Explain how the DIG-labeling and detection kit that you used works.
10. When doing a northern blot, why do you avoid soaking the gel in 0.5 M NaOH/1.5 M
NaCl as you do in Southern blotting?
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