MCB 7300
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 procedure most
commonly used for this is called "Southern gel blotting" and is followed by hybridization
analysis.
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MCB 7300
Dr. Showalter
Southern Blot Lab: Overview
Day
Gel hybridization
Jan. 15
Genomic DNA isolation
(Monday)
PCR amplification (TA)
Jan. 22
Electrophoresis & transfer
(Wednesday)
Colony Hybridization
Genomic DNA isolation
PCR amplification (TA)
Ligation and transformation
Jan. 23
(Thursday)
Membrane cross linking and
membrane stored at 4°C
Plates stored at 4ºC
Jan. 27
(Wednesday)
-
Colony lifting
March 17
(Monday)
Probe labeling and overnight
incubation at 37°C; probe
stored at -20°C (TA)
Prehybridization and
incubation for overnight
Adding probe and overnight
incubation at the hybridization
temperature
Washes and detection
Probe labeling and overnight
incubation at 37°C; probe
stored at -20°C (TA)
Prehybridization and
incubation for overnight
Adding probe and overnight
incubation at the hybridization
temperature
Washes and detection
March 18
(Tuesday)
March 19
(Wednesday)
Materials needed:
0.2 N HCl
0.5 M NaOH/1.5 M NaCl
1.5 M NaCl/0.5 M Tris-HCl, pH 7.4
Nitrocellulose membranes
Paper towels
Glass plate
Pyrex dish
Tupperware dish/or sealable bag
DIG High Prime labeling and detection kit
20X, 2X SSC
0.2M EDTA (pH 8.0)
Whatman 3MM filter paper
Sponge
Weight
Shaking incubator or water bath, temperature controlled
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Dr. Showalter
Southern Blot Lab: Detailed Protocol
Day 1
A. Genomic DNA extraction, PCR amplification and gel electrophoresis
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.
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 or nylon membrane (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
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.
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Dr. Showalter
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.
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 (either the PCR amplified fragment or plasmid
harboring the same fragment cloned in bacteria) and sterile, double distilled
water to a final volume of 24 l.
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Dr. Showalter
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. (15l of probe for colony lift and 15 l for the
DNA Southern blot lift).
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 or petri dish). Add enough prehybridization solution (i) to fully
cover the membrane and prehybridize overnight, 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.
Calculating the optimal hybridization temperature
The appropriate hybridization temperature is calculated according to GC content and
percent homology of probe to target according to the following equation:
Tm = 49.82 + 0.41 (% G + C) - (600/l) [l = length of hybrid in base pairs]
Toptimal = Tm - 20 to 25° C
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Dr. Showalter
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 an Arabidopsis mutant (Columbia line). An
Arabidopsis gene has been amplified using gene specific primers. This is the PCR
product that was cloned into the TOPO vector. 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. The PCR product was run as control for the experiment.
For the colony lift, the transformed E. coli cells 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 T-DNA 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 I (Cat. No. 1 745 832 910) and Roche DIG High Prime
DNA labeling and Detection Starter Kit II (Cat. No. 11 585 614 910).
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Dr. Showalter
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 March 26, 2014):
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 a hard
copy of their lab report.
 Abstract
 Introduction (on the method and objectives and the gene you amplified)
 Results
 Discussion
 Materials and methods (summarized protocol)
 Acknowledgements
 References
 Answers to the following Questions:
1. Explain how the digoxigenin (DIG) DNA labeling and detection kit that you used
works? In other words, explain how DIG labeling and CSPD detection is done.
2. Explain how digoxigenin (DIG) DNA labeling and BCIP/NBT detection is similar
and yet different from your answer to question 1.
3. Explain another way to label a DNA probe for Southern hybridization analysis?
4. Compare and contrast Southern blotting to northern blotting and microarray analysis.
5. What is the purpose of prehybridization?
6. What two hybridization parameters or conditions are typically altered for maximum
hybridization?
7. What are zoo blots and garden blots?
8. What kinds of nucleic acid bind to nitrocellulose? What kinds of nucleic acid do not
bind?
9. Why do you soak the gel and not the colony lift in 0.2N HCl?
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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