Southern Transfer and Hybridization
Gel Electrophoresis and Southern Transfer to Nitrocellulose (Part 2)
Molecular Biology Lab #8
Background:
Southern transfer and hybridization is a method for detection and analysis of single genes
in a complex mixture of high molecular weight DNA. There are many applications of this
method such as molecular diagnostics in medicine or forensic science. DNA finger
printing is a form of Southern hybridization. For example, an individual might inherit one
copy of a disease susceptibility gene from one parent and this might predispose to the
disease in later life. To determine whether the individual is at risk or not, a sample of
DNA from both parents and offspring could be digested with a restriction endonuclease
and the resulting fragments separated by gel electrophoresis. The gene could be probed,
after Southern transfer to a membrane, in order to examine its restriction pattern. If the
pattern matched the affected parent, the individual has the susceptibility gene. A similar
scenario is used in forensic science to match a suspect’s blood to any body tissue or
contaminating cells left at the crime scene.
In Southern transfer, the agarose gel containing thousands of restriction fragments is
blotted onto a nylon or nitrocellulose membrane. Usually, this is done in several steps.
First, the DNA within the gel is denatured by placing the gel in alkali with 1.5 M NaCl.
The alkali causes the DNA strands to denature and the salt allows the DNA to become
mobile. After denaturation, the pH is neutralized and the gel is placed on a solid support.
The high salt solution is drawn through the gel from below by capillary action and it
carries the DNA with it to deposit it on the overlying membrane. This creates an exact
replica of the DNA within the gel on a solid support. One specific gene can be detected
from all the others by hybridization of a denatured probe DNA to the denatured DNA on
the filter.
Objectives:
The objectives of this lab are to perform gel electrophoresis to separate restriction
fragments of high molecular weight DNA, and then to use capillary blotting (Southern
transfer) to transfer the restriction fragments to a nitrocellulose membrane.
Materials:
Ethidium bromide staining solution (0.5% ethidium) 1 liter
UV illuminator
Denaturation solution (1.5 M NaCl and 0.5 M NaOH, 2 liters)
Neutralization solution (1 M Tris and 1.5 M NaCl, 3 liters)
4 packs of paper towels
4 large glass baking dishes
Parafilm
4 scalpels or razor blades
Blunt end forceps
Whatman 3M chromatography paper
20X SSC (8 liters)
6X SSC (1 liter)
Nylon membranes
500 g weight for transfer
UV cross linker
1X TAE gel running buffer
Agarose
Micropipetters, yellow tips
Plastic burger flipper
Large agarose gel electrophoresis box
Power packs
Gel loading buffer (6X)
Restriction digested DNA samples
1 kb molecular weight markers
Methods:
1. Load samples from the restriction digest into wells of a large 0.8% agarose gel. If
you precipitate your restriction digest with salt and ethanol, be sure to remove all
traces of ethanol from your eppendorf tube. Place the tubes containing
resuspended DNA at 60ºC for 10 min to evaporate any residual ethanol. Ethanol
can cause problems when you load your samples into the wells.
2. Run the gel briefly at 90V to drive DNA samples into the agarose. Turn down the
voltage to 20 V overnight and allow to run.
3. Stop the gel after 12 hours. The blue front of bromophenol blue dye should have
migrated about 70-80% of the length of the gel.
4. Place the gel in an aqueous solution of ethidium bromide (0.5%) and rock gently
on a shaker platform for 15 minutes to stain the DNA.
5. Remove the ethidium bromide and replace with water to destain the gel for 10
min. The gel should have a smear of DNA corresponding to thousands of
restriction fragments of different sizes. Photograph the gel for your record.
6. Cut off any portion of the gel that is not being used. Trim the top of the gel away,
leaving a little of the wells so that you can orient the gel. Trim one or more
corners of the gel so that you can distinguish it from the gels of other groups.
7. If you are interested in examining large DNA fragments, you should treat the gel
with 0.2 N HCL to depurinate the DNA and facilitate movement of the DNA from
gel to nitrocellose. In this experiment, the DNA is smaller and depurination is not
necessary.
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8. Transfer the gel to denaturation solution for 45 min using constant agitation on a
slowly rocking platform. Use at least 10 gel volumes of denaturation solution to
rinse.
9. Rinse the gel briefly in deionized water to remove surface alkali. Place the gel in
neutralization buffer for 30 min with gentle rocking. After 30 min, transfer to
fresh neutralization buffer for 15 min.
10. Prepare the nylon membrane for transfer. Measure the length and width of your
gel. Cut a piece of nylon using a ruler and scalpel or sharp razor. The size of the
nylon filter should be about 1-2mm larger than the underlying gel. Do not touch
the nylon with your bare fingers because you will transfer oil from your skin.
Wear gloves. Handle the membrane by its edges using forceps.
11. Place the membrane into distilled or deionized water to rehydrate (it should
quickly turn darker gray). If the membranes fail to wet completely after several
minutes, discard. Allow to wet for 5 min.
12. Place 1 liter of 10X SSC in a large glass baking dish. Place a solid support (such
as an inverted gel form in the bottom of the dish to support the gel.
13. Cut pieces of 3M chromatography paper about 8 inches wide and 10 inches long.
Lay the stack of 3 to 4 chromatography paper towels directly over the plastic gel
form. Allow the ends to be submerged in the 10X SSC. Using a glass pipette,
gently roll out any air bubbles that are present under the 3M papers. The 3M
paper forms a wick that carries 10x SSC up to the gel.
14. Place the gel directly on top of the wet 3M filter paper. Gently add some 10X
SSC to the top of the gel and roll out any air bubbles between the gel and the
underlying paper using a glass pipette. Air bubbles will completely block transfer
of DNA and will make spots on your final hybridization experiment.
15. Place the nylon membrane over the top of the gel. Add some 10X SSC and use a
pipette to gently smooth out any air bubbles. Once the nylon has been positioned,
do not to move it again.
16. Place pieces of parafilm tightly around the edges of the gel. This serves to prevent
the paper towels from sagging and touching the wick. This would be a problem
because it would short circuit the fluid flow around the gel rather than through it.
It would cause poor transfer.
17. Place several pieces of 3M paper or thicker blot paper over the nylon. Wet the
layers with 10X SSC and then roll out air bubbles with a glass pipette.
18. Stack paper towels on top of the gel to about 4 inches in height. Place a weight of
approximately 500 grams on top of the paper towels.
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19. Allow the capillary transfer to run overnight. The salt solution should carry all of
the DNA from the gel to the underside of the filter. The next day, the paper towels
should be wet from the fluid sucked out of the baking dish. Place enough 10X
SSC in the baking dish (1 liter) so that it is not totally depleted.
20. After the transfer is complete, remove the paper towels down to the level of the
membrane. Carefully remove the membrane with forceps and place in 6X SSC on
a shaker for 5 minutes. This washes away any agarose that might stick to the
membrane.
21. After washing, place the membrane on paper towels and allow to become semi
dry (5-10 min). Place the membrane in a UV cross linker and irradiate with 1.5
J/cm2 to cross link DNA to the membrane.
22. Store the membrane at 4ºC until you are ready to make probe and hybridize.
23. Clean up your work area and wipe up any spills!
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