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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 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 0
Jan. 14
(Wednesday)
Day 1
Genomic DNA Isolation
PCR Amplification (TA)
Gel Electrophoresis
Feb. 23
(Monday)
Transfer to Nitrocellulose Membrane
Day 2
Feb. 24
(Tuesday)
Day 3
Feb. 25
(Wednesday)
DIG-DNA Labeling
DNA Fixation
Hybridization
Need 1 hr with TA in Porter 504 (10AM)
Immunological Detection
Materials needed:
0.2 N HCl
0.5 M NaOH/1.5 M NaCl (Denaturation solution)
1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 (Neutralization solution)
Nitrocellulose membranes
0.2M EDTA (pH 8.0)
20X and 2X SSC
DIG High Prime DNA Labeling and Detection Starter Kit I
Paper towels
Glass plate
Pyrex dish
Tupperware dish or sealable bag
Shaking incubator or water bath, temperature controlled
Whatman 3MM filter paper
Sponge
Weight
The protocol for this lab was adapted from Roche DIG High Prime DNA
labeling and Detection Starter Kit I (Cat. No. 11 745 832 910) and Roche DIG High
Prime DNA labeling and Detection Starter Kit II (Cat. No. 11 585 614 910).
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Southern Blot Lab: Detailed Protocol
Day 0 Genomic DNA Isolation 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 Arabidopsis Genomic DNA (25-100ng/l)
2.0 l 10X buffer
0.25 l dNTPs
0.25 l Taq DNA polymerase
1 l Forward primer for GALT6 (At5g62620) [10 M]
1 l Reverse primer for GALT6 (At5g62620) [10 M]
Add water to 20 l total volume
Day 1 Gel Electrophoresis, Transfer to Membrane and DIG-DNA Labeling
Step 1: Gel electrophoresis
Genomic DNA and PCR product along with the 2-Log DNA ladder (0.1-10 kb) marker
[NEB catalog #N3200S] run on a mini-gel for 1.5- 2h. (Lanes: DNA marker, Arabidopsis
genomic DNA digested with Hind III, genomic DNA GALT6 PCR product, pEarleyGate
101-GALT6 PCR product)
Step 2: Denaturing double-stranded DNA in the gel
1. Place gel in a “tip box” and add 0.2 N HCl for 10 min
2. Wash the gel with water, 3 times
3. Add Denaturation solution (0.5 M NaOH/1.5 M NaCl), twice for 15 min.
4. Add Neutralization solution (1.5 M NaCl/0.5 M Tris-HCl, pH 7.4). Incubate
for 30 min and rinse with water
Step 3: Setting up the gel for transfer to nitrocellulose membrane
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
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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 membrane 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. Use some tape to stabilize the setup. Cover the
edges of the tray with plastic wrap. Allow the transfer to proceed overnight.
Step 4: DIG-DNA labeling (Make the probe).
1. 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 16 l. Note that we are using the GALT6 PCR
product from the plasmid as a probe.
2. Denature the DNA by heating in a boiling water bath for 10 min and quickly
chilling in an ice/water bath.
3. Mix DIG-High prime (vial 1) thoroughly and add 4l to the denatured DNA,
mix and centrifuge briefly.
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4. Incubate overnight at 37oC
5. Stop the reaction by adding 2 l 0.2M EDTA (pH 8.0) or heating to 65oC for
10 min (will be done by TAs).
Day 2 DNA Fixation and Hybridization
Step 1: DNA fixation
1. Take apart the transfer setup and rinse the membrane in 2X SSC for 5 min.
2. Place the membrane on Whatman 3MM-paper soaked with 10X SSC.
3. UV cross-linking for 1 min to covalently link the DNA to the membrane.
This membrane is now ready for hybridization.
Step 2: 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.
1. Preheat an appropriate volume (10ml/100 cm2 membrane) of prehybridization
solution (DIG Easy Hyb) to the appropriate hybridization temperature (to be
announced).
2. Place the membrane in a sealable bag or plastic container (Tupperware or petri
dish). Add enough prehybridization solution to fully cover the membrane and
prehybridize for 30 min, shaking gently, at the hybridization temperature.
3. Denature DIG-labeled probe by boiling for 5 min and rapidly cooling in an
ice/water bath.
4. Add 20 l denatured DIG-labeled probe to the preheated DIG Easy Hyb and
mix well (but gently to avoid bubbles that increase background).
5. Pour off the prehybridization solution and add probe/hybridization mixture to
the membrane.
6. Incubate 4 h to overnight at the hybridization temperature with gentle agitation.
Calculating the optimal hybridization temperature
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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
Note GALT6 probe is 2,040 bp and 46% G+C
Day 3 Immunological Detection
Step 1: Stringency washes
1. Rinse the membrane in 2X SSC, 0.1% SDS, for 5 min at RT with constant
agitation (2 times).
2. Rinse the membrane in 0.5X SSC, 0.1% SDS (pre-warmed to 65oC) at 65oC for 15
min (2 times).
Step 2: Detection and detection
1. Rinse the membrane briefly (1-5 min) in washing buffer.
2. Incubate the membrane in 100 ml blocking solution for 30 min.
3. Incubate the membrane in 20 ml antibody solution for 30 min.
4. Wash the membrane in 100 ml washing buffer for 15 min (2 times).
5. Equilibrate the membrane in 20 ml detection buffer for 2-5 min.
6. 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 hr).
7. Stop color development, when the desired band intensities are achieved, by
washing the membrane for 5 min with 50 ml sterile double distilled water.
8. Results can be documented by photocopying the wet membrane or by photography.
Conclusions:
Did the probe recognize the genomic DNA from the gel? If so, what does that mean?
If not, why not?
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Lab Report (100 points, due date to be announced):
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 BCIP/NBT detection is done.
2. Explain how the digoxigenin (DIG) DNA labeling and CSPD 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 a gel and not a 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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