SOUTHERN BLOT LABS: BLOTTING AND HYBRIDIZATION OF COLONY
LIFTS AND AN AGAROSE GEL WITH DIGESTED GENOMIC DNA
Hybridization Analysis: Detection of Specific DNA Sequences
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 us
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 such application is the screening of DNA libraries. A 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 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 these is
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called "Southern blotting" (named after the man who invented it) and is followed by
hybridization analysis.
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
Day 1
A. Digesting genomic DNA (TAs prepared this)
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, run on a mini-gel for 1.5-2 h
B. 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
C. 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.
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iv. Place the membrane, colony-side up, onto filter paper saturated
with 0.5 M NaOH/1.5 M NaCl. 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. 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.
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. Incubate twice for 15 min.
iii. Place gel in 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4. Incubate for 30
min.
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.
Setting up the gel for transfer (i.e., blotting)
Prepare the transfer setup: Place a sponge in the bottom of a glass tray and fill the dish half
way with 20X SSC. 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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Day 2
D. 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 (to be done on Tuesday
between noon and 3pm)
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.
Day 3 Washes and detection
E. 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
F.
i.
ii.
iii.
iv.
v.
vi.
vii.
viii.
Detection
Rinse the membrane briefly (1-5 min) in Washing Buffer
Incubate for 30 min in 100 ml Blocking solution.
Incubate for 30 min in 20 ml Antibody solution
Wash 2 X 15 min in 100 ml Washing buffer
Equilibrate 2-5 min in 20 ml Detection buffer.
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).
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.
Results can be documented by photocopying the wet membrane or by
photography.
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Conclusions:
Did the probe recognize any DNA fragments (or colonies) from the gel? If so, what does that
mean? If not, why not?
Important Notes:
Genomic DNA of Arabidopsis thaliana was digested with either BamHI or HindIII. There
was control DNA from wildtype (Col-0) and mutant plants, as well as the DNA of two plants of
unknown genotypes. The mutant line is the line SALK_066961 and the unknowns are offspring
from crosses between wildtype and mutant. Each student will have 4 DNA samples, one
wildtype, one mutant and two unknowns, which were all cut with either BamHI or HindIII.
Plasmid DNA (from lab 3, Dr. Wyatt’s lab) will be used as positive control DNA. The PCR
product that was cloned into the TOPO vector (lab 2, Dr. Wyatt’s lab) will be used as template to
make the probe. A map of that gene (At5G67230) was given to the students earlier. Using the
band patterns from the wildtype and mutant plants, the students have to identify the genotype of
the unknowns.
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 did not contain X-Gal and IPTG. The cells
contain the TOPO vector into which a part of At5G67230 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 DIG High Prime DNA labeling and Detection Kit
1 (Cat. No. 1 745 832) Roche Scientific.
Lab Report (100 points, due on Monday, May 4th at the beginning of lab):
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 are the sizes of the fragments to you expect to get with your digested DNA if it is a
wildtype, homozygous mutant or heterozygous mutant plant? The gene ID is At5G67230
(IRX14) and the mutant line used is SALK_066961. (Hint: start at the TAIR webpage
www.arabidopsis.org).
7. What is a zoo blot and a garden blot?
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. Explain how the DIG-labeling and detection kit that you used works.
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