7. Bake this filter paper and membrane “sandwich” for 30–60 min in a 70–80°C oven.
8. The membrane may be stored indefinitely at room temperature.
F. Prehybridization
1. With gloved hands or with blunt forceps, place the membrane all the way down into one of the hybridization bags provided. Handle the membrane gently and only by the corners.
2. Pour or pipet 10 mL of Prehybridization Buffer into the bag. Starting at the bottom, squeeze the bag gently to push most of the bubbles toward the top. The goal is to have a thin layer of fluid covering the membrane; air bubbles can prevent contact between the membrane and the fluid. When most of the bubbles have been squeezed past the zip-lock strip (expect some buffer to be lost), carefully seal the bag. Make certain that the bag is sealed.
3. Tape the top of the bag to the inside wall of a 50°C water bath, allowing the membrane-containing portion of the bag to hang in the water. If there are still bubbles in the bag, move them to the area of the bag above the membrane.
4. Incubate 90 min to overnight.
G. Hybridization
1. Remove the bag from the water bath, open the bag, and pour out the Prehybridization Buffer. Immediately add 10 mL of Hybridization Buffer. (Hybridization Buffer has the same composition as Prehybridization Buffer, except that the biotinylated oligonucleotide probe has been added.) Do not allow the membrane to dry out at all between when the Prehybridization Buffer is removed and the Hybridization Buffer is added. Make sure that the probe has been added to the Hybridization Buffer before you pour off the Prehybridization Buffer.
2. Work bubbles out of the bag, as before.
3. Place the bag in the water bath, as before.
4. Hybridize overnight.
H. Washing and probe detection (color development) Note:
Follow these directions carefully. Do not reduce the Wash Buffer volumes. Do not allow the membrane to dry out between buffer changes.
1. Place 100 mL of Wash Buffer into a plastic container with a tight-fitting lid.
2. Remove the nylon membrane from the hybridization bag and immediately place it in the Wash Buffer in the plastic container. Discard the bag and Hybridization Buffer. Agitate the membrane very gently for 5 min at room temperature (it should not bend or tumble).
3. Pour off the Wash Buffer, immediately add 100 mL of fresh Wash Buffer, and agitate gently for 5 min.
4. Pour off the Wash Buffer, add 100 mL 50°C Wash Buffer, and agitate gently for 5 min. If possible, float the container (with the lid on) in a 50°C water bath during this wash step, to maintain temperature.
5. Pour off the Wash Buffer, immediately add 100 mL of Buffer 1 to the container, and agitate gently for 5 min at room temperature.
6. Pour off Buffer 1, add 100 mL of Buffer 2, and agitate gently for 30 min at room temperature. Do not reduce the agitation time.
7. Pour off Buffer 2, place the membrane (with the side labeled DNA facing up) in a clean weigh boat, and immediately add 10–15 mL of streptavidin-alkaline phosphatase conjugate in Buffer 2. Then, rock the membrane very slowly at room temperature for 10–15 min. The solution should move quite slowly across the membrane.
8. Place the membrane back into the plastic container, immediately add 100 mL of Buffer 1, and agitate the container gently for 15 min at room temperature (it should not bend or tumble). Rinse and dry the weigh boat.
9. Pour off Buffer 1, immediately add 100 mL of fresh Buffer 1, and agitate the container gently at room temperature for 15 min, as before.
10. Remove the membrane and place it (with the side labeled DNA facing up) in the clean weigh boat. Immediately add 50 mL of Buffer 3 and let it stand for 5 min at room temperature.
11. Pour off Buffer 3, immediately add 15 mL of fresh NBT/BCIP color development solution, and place the weigh boat in a dark place such as a cabinet or drawer. Color development will require 30 min to 2 hr; check the membrane periodically, but do not move it. When the bands have appeared and darkened, pour off the color development solution and replace it with distilled or deionized water. Let the membrane stand in water for 5 to 30 min, then remove the membrane and allow it to air dry. Store the membrane in a dark place, as light will fade the bands.
Analysis of Results
The oligonucleotide probe is a 21-base sequence from the bacteriophage lambda genome. The data from this exercise allows students to determine the approximate location of that sequence within the lambda genome.
First, students need restriction maps of lambda for the three enzymes, HindIII, EcoRI, and BstEII. The restriction site locations for these enzymes are listed below. Have students draw maps from this data and determine the fragment lengths that would result from digestion of lambda DNA with these enzymes.
Next, students must determine which bands from each of the digests hybridized to the probe. To do this, they must measure the distance between the marks on their membrane (showing the locations of the wells) down to the band that developed after hybridization. Use this measurement and similar measurements made using the photograph of the gel with the ruler to determine which bands on the gel correspond to the bands that develop on the membrane after hybridization. Use the ruler in the photograph to measure the gel in the photograph. There can be some error in this process because the pencil marks indicating the well location may not line up exactly with the location of the wells. In addition, the transfer may not have been exactly vertical if the transfer stack was off balance.
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