90-minute Classes (Experiment can be completed in 5 days.)
3-hour Laboratory Periods (Experiment requires three labs.)
Preparation
Southern hybridization analysis and nonradioactive detection require many different solutions. We have supplied concentrated stock solutions for preparing them; please follow the instructions carefully. It may take several hours to prepare all the solutions. Note: To save you money, we have not included much excess solution of any kind. It is important that students use the recommended amounts of solutions. After preparing the Prehybridization Buffer, Hybridization Buffer, and Buffer 2, store them in the refrigerator. Store other solutions at room temperature unless it is otherwise stated.
General
1. Denaturation Buffer: Dilute 500 mL of 2× denaturation buffer to 1 L of 1× with distilled or deionized water.
2. Neutralization Buffer: Dilute 500 mL of 2× neutralization buffer to 1 L of 1× with distilled or deionized water.
3. Buffer 1: Dilute 150 mL of 2× neutralization buffer with 2850 mL distilled or deionized water to make 3 L of Buffer 1.
4. 10× SSC: Dilute 1 L of 20× SSC with 1 L of distilled or deionized water to make 2 L of 10× SSC.
5. Wash Buffer: Add 200 mL of 20× SSC to 1780 mL of distilled or deionized water, plus 20 mL of 10% SDS, to make 2 L of wash buffer.
6. 2× SSC: Dilute 100 mL of 20× SSC with 900 mL of distilled or deionized water to make 1 L of 2× SSC.
7. Prehybridization Buffer and Hybridization Buffer: Hybridization Buffer has the same composition as Prehybridization Buffer, except that Hybridization Buffer has biotinylated oligonucleotide probe added. Dissolve 2.5 g of blocking agent in 250 mL of Prehybridization Buffer.Stir well. The solution will be white, but no particulate matter should remain. Remove 100 mL to a separate, clean container marked Hybridization Buffer, 100 mL. Store both containers in the refrigerator until use. Just before the hybridization (Step G), add 30 μL of biotinylated oligonucleotide probe to the 100 mL solution to make the Hybridization Buffer.
8. Buffer 2: Dissolve 7.5 g of blocking agent in 750 mL of Buffer 1 (made in Preparation Step 3). Stir well. The solution will become white, but no solid particles should remain. Remove 100 mL of the Buffer 2 that you just made to a clean container labeled 100 mL of Buffer 2 for SA-AP.
The 100 μL streptavidin-alkaline-phosphatase conjugate will be added to this 100 mL on the day of use (Step H7). Store both containers in the refrigerator until use.
9. Buffer 3: Use as supplied.
10. Prepare membranes: While wearing gloves, cut each 10- × 15-cm nylon membrane into two 10- × 7.5-cm membranes. Handle the membranes by the corners and edges. Do not fold, rub, or crinkle them. Replace them between their paper liners and put them back into the zipping bag.
Daily preparation
Before Step A, preparing and running a 1.0% agarose gel
• Depending on your schedule, you may need to cast the gels in advance so that students can immediately load their samples.
• Each group will need:
1 1.0% agarose gel
1 sample each: λ cut w/EcoRI, λ cut w/HindIII, and λ cut w/BstEII DNA equipment for loading the gel
• The photography station should also have a small, transparent, metric ruler. Students must lay the ruler on the edge of the gel so that, from the photograph, they can tell the distance that each individual DNA band has migrated away from the gel wells.
Before Steps B, C, and D, transfer procedures
• Each group will need:
1 plastic container with a tight-fitting lid
150 mL denaturation buffer
150 mL neutralization buffer
1 weigh boat
300 mL 10× SSC
1 7.5- × 10-cm nylon membrane
1 sheet Whatman 3MM filter paper
1 pencil
1 pair of gloves
scissors
Parafilm® or plastic wrap
brown paper towels
100-mL graduated cylinders
a means to measure 10 mL
1 400-mL beaker
1 flat piece of plastic or glass, 8 × 10 cm or slightly larger
Before Step E, taking down the stack, rinsing the membrane, and baking the gel
• Preheat oven to 70–80°C.
• Each group will need:
1 plastic container with a tight-fitting lid
100 mL 2× SSC
2 approximately 11- × 12-cm sheets of Whatman paper (cut 6 from each of the two remaining pieces of Whatman 3MM paper)
Before Step F, prehybridization
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