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As a first step in the hybridization procedure, the membrane is immersed in a prehybridization buffer that prevents the probe from binding to the membrane in a nonspecific manner. After this prehybridization step, the membrane is transferred to the hybridization solution containing the labeled probe. The composition of the hybridization solution and the hybridization conditions vary depending upon the probe used and the DNA sequence you wish to detect. After the hybridization is finished, the membrane is rinsed repeatedly under conditions that will remove unhybridized and nonspecifically bound probe, but that will not disrupt hydrogen bonds between the probe and the target sequence in the sample DNA.
The final step in Southern hybridization is to detect the hybridized probe. In this kit, the probe is attached to a molecule called biotin. To detect this biotin-labeled probe, the hybridized membrane is soaked in a solution containing a two-component molecule. One component is streptavidin, a molecule that binds tightly to biotin. The other component is the enzyme, alkaline phosphatase. The alkaline phosphatase protein and the attached streptavidin together are called a conjugate, or a protein conjugate. During the time that the membrane is soaked in the solution containing the streptavidin-alkaline phosphatase conjugate, the streptavidin binds tightly
to the biotinylated probe. The alkaline phosphatase becomes attached to the probe by virtue of its bond to streptavidin. Once the incubation step to bind the streptavidin-alkaline phosphatase conjugate to the biotin-labeled probe is finished, the membrane is rinsed to remove the unbound conjugate.
Finally, the membrane is placed in a color development solution containing two components: 5-bromo-4-chloro-3-indolyphosphate (BCIP) and nitro blue tetrazolium (NBT). The alkaline phosphatase portion of the conjugate removes a phosphate group from BCIP; the resulting product dimerizes to form a dark blue precipitate. The dimerization reaction also releases hydride ions that reduce the NBT; the reduced NBT forms a purple precipitate. Since the alkaline phosphatase is bound to the probe via its connection to streptavidin, the precipitates from its reaction with BCIP and NBT form where the probe is bound to the membrane, thereby indicating the location of the DNA fragments hybridized to the probe.
Student Preparation
Before attempting this exercise, students should be familiar with the theory of restriction enzyme analysis and the mechanics of running gels. They should have been introduced to the concepts of hybridization analysis, as well. Further information on hybridization analysis as well as paper-and-pencil exercises that illustrate the concepts can be found in Recombinant DNA and Biotechnology: A Guide for Teachers by Kreuzer and Massey (2001; ASM Press, Washington, DC; Carolina Biological Supply catalog #RN-21-2218).
Materials
Included in the kit
12 staining trays (weigh boats)
12 pairs gloves
3 10- × 15-cm positively charged nylon membranes
8 sheets Whatman 3MM filter paper
8 hybridization bags
100 mL bottle NBT/BCIP color development solution
6 Student Guides
Needed, but not supplied:
electrophoresis equipment for 6 groups
1.0% agarose gel, electrophoresis buffer, and stain for 6 gels
6 small, transparent metric rulers
6 pencils
6 scissors
stacks of brown paper towels
6 plastic containers with tight-fitting lids
6 shallow containers, approximately 28 × 51 cm, for transfer
6 400-mL beakers (1 per group)
6 flat pieces of plastic or glass, 8 × 10 cm or a little larger
Parafilm® or plastic wrap
6 100-mL graduated cylinders
distilled or deionized water
water baths
gel photography equipment
access to oven (80ºC)
Scheduling
There are several steps to Southern hybridization analysis. Letters correspond to the steps of the laboratory activities in this kit.
A. Running, staining, and photographing the gel.
The time this step requires depends upon the staining system used. The gel takes about 1 hr to run at 130 V and must be photographed before treatment for the transfer. Ethidium bromide is the fastest stain, requiring only 15 min. If CarolinaBLU™ is incorporated into the gel (this does not interfere with hybridization analysis), the gel can be stained for only 15 min and destained for 15–30 min with continuous changes of water.
B. Treating the gel (denaturation) and setting up the transfer stack.1 hr for treatment and 10–15 min to assemble the stack
C., D. Overnight transfer.
E. Washing and baking the membrane.30 min for washing and a minimum of 30 min for baking
F., G. Prehybridizing and hybridizing the membrane. Prehybridize 90 min to overnight; hybridize overnight
H. Washing the membrane and developing the color.70 min for the washes and color development; 1–2 hr for bands to develop
Suggested schedules
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