Southern Hybridization Experiment Kit

Before Step G, hybridization
• Prepare the Hybridization Buffer. Add 20–30 μL biotinylated probe(contents of the tube) to the set-aside container labeled Hybridization Buffer, 100 mL from Preparation Step 7.
• Each group will need:
10 mL Hybridization Buffer

Before Step H, washing and probe detection (color development)
• So that students can perform the third wash step at 50ºC, pre-warm 600 mL of Wash Buffer to 50ºC (42ºC is adequate, if this is as warm as your bath gets). Pre-warm the buffer by placing a closed container holding 600 mL of buffer into a water bath. A tightly closed container of Wash Buffer can be left in a water bath overnight.
Students can perform the third wash step by floating the containers, with the lids on, holding the membranes and pre-warmed buffer in the water bath during the wash. Another way to set this up is to fill a few insulated foam containers with 50ºC water just prior to use. The hot taps of many sinks provide water that is close to 50ºC.
• Prepare streptavidin-alkaline phosphatase (SA-AP) conjugate solution just before use. On the day you are to use it, add 100 μL (contents of the tube) of SA-AP conjugate to the 100 mL of Buffer 2 set aside and labeled 100 mL of Buffer 2 For SA-AP from Preparation Step 8.
• Each group will need:
1 100-mL graduated cylinder for measuring buffer volumes
200 mL wash buffer at room temperature
100 mL wash buffer at water bath temperature (42–50ºC)
300 mL Buffer 1
100 mL Buffer 2 (can be put at room temperature for the lab period)
10–15 mL SA-AP solution
50 mL Buffer 3
15 mL NBT/BCIP color development solution
(solution comes ready-to-use)
a clean weigh boat
gloves

Note: To save you money, we have not included much excess material in this kit. It is important that students use the recommended amounts of solutions.

Laboratory Procedures
A. Prepare and run a 1.0% agarose gel
1. Each student group should have (or prepare) a 1.0% agarose gel.An 8- × 10-cm gel requires 50 mL of agarose solution. For 1.0% agarose, use 0.5 g of agarose per 50 mL of 1× TBE buffer.(CarolinaBLU™ Gel and Buffer Stain may be incorporated into the gels and the TBE electrophoresis buffer to decrease the staining time. Follow the instructions included with the stain.)
2. Noting the order, load the three DNA samples in adjacent lanes; if possible, leave an outside lane empty so that the gel can be trimmed if it is more than 7.5 cm wide. The entire contents of each sample tube should be loaded in a well. (This amount of DNA will look overloaded if stained with ethidium bromide, but works well for the transfer and detection.)
3. Run the bromophenol blue dye to the bottom of the gel. This takes about 1 hr at 130 V.
4. Use CarolinaBLU™ or other stain of your choice to visualize the DNA. The gel must be photographed alongside a ruler before treatment for transfer. Lay a transparent ruler on the gel so that you can determine the distance from the wells to a particular DNA band from the photograph (Figure 1).

Figure 1.

5. Gels can be stored refrigerated overnight in 1× TBE buffer before transfer, if necessary. Refrigerating the gels during any overnight storage will help prevent the DNA bands in the gel from diffusing.

C. Prepare materials for the transfer stack
1. Prepare the membrane.
Note: The positively charged nylon membrane is vulnerable to abrasion and grease. Wear gloves, and handle it by the corners at all times. Do not bend or abrade it. Use a pencil to write DNA and your group’s initials in small letters at the center of the 7.5-cm side of the 7.5- × 10-cm membrane. (The side you write on will contact the gel.) Put a small amount of deionized or distilled water in your weigh boat and float the membrane on the water until it is thoroughly wet. Then, remove the membrane from the water, replace the water with a small amount of 10× SSC, and float the membrane on the solution. Leave it there until you assemble the transfer stack.