6. SOUTHERN HYBRIDIZATION
This is a technique of detecting DNA by using a DNA probe. In this technique, DNA is separated by gel electrophoresis and then transferred from the gel to a membrane by blotting. The DNA was detected from the membrane with a DNA probe to complementary bind to DNA.The probe was labeled by Alkaline phosphatase, that was observed by chemical luminescence that was observed as a glowing light.Digesting DNA with restriction enzymes, EcoRI was used, which recognizes the sequences of GAATTC and specifically cuts between G and A, and A and G of the complementary sequence.
In the experiments carried out, 2 fragments of 950 bp and 4550 bp respectively, were obtained.
Agarose gel electrophoresis: the agarose gel electrophoresis was prepared and a 6-well gel (for a larger volume) was used. 40 µl of sample and 8 µl of 6 x loading buffer (blue color) was loaded into each of the wells giving a total sample of 48 µl. 5 µl of 50ng/µl lambda Hind III was used as the molecular marker while the loading buffer used was 1 x TAE.
The DNA band was cut out of the gel under UV light and its weight taken. This was done by taking the weight of an empty eppendorf tube and repeating with the gel slice containing the DNA sequence inserted into the tube, and the difference calculated to obtain the actual weight of the gel slice. For each 100 mg of gel slice, 300 µl of buffer QG was added, hence for 178.24 g, 535 µl of the buffer was added. A second gel electrophoresis was carried out again.
Samples were loaded and ran for 30 minutes. The DNA was determined by using a restriction enzyme lambda Hind III for digestion.
Membrane neutralization: Pieces of positively charged nylon membranes were cut into 1 mm, and 6 sheets of thick blotting paper cut to the same size as that of the membrane. The corner of the membrane was cut to match that of the cut corner of the gel. 2 pieces of thick blotting paper were soaked with alkaline Denaturation solution on glass plate to form the support. The blotting paper was made wet with alkaline Denaturation solution and 3 pieces of blotting paper placed at the center of the support paper on the glass plate. Any bubbles that formed between the papers were removed. The top of the gel was made wet with alkaline Denaturation solution and wet membranes placed on top. A stack of paper towels (about 5 cm thick) were placed on the blotting papers and a glass plate placed on top of the stack and weighed down with a 300-g weight. This position was left for 8-24 hours to allow DNA to transfer, and paper towels were subsequently replaced as they became wet with buffer.
Hybridization: the paper towels and blotting papers were removed from above the gel, and gel laid upside down on a dry sheet of blotting paper. The positions of the gel slots were maked on the membrane using a soft lead pencil. The gel was peeled off from the membrane and discarded. The membrane was placed on the blotting paper soaked with neutralization buffer for 5 minutes; afterwhich it was placed on a new Saran wrap, and subsequently dried using a hair drier. Irradiation at 254 nm was done to cross-link the DNA to the membrane, and then wrapped loosely with aluminium foil or blotting paper. The membrane was finally stored at room temperature.
Preparation of labeled probe: 20 µl of crosslinker solution was diluted with 80 µl of water. DNA to be labeled was diluted to 10ng/µl concentration and 10µl of the diluted DNA solution placed in a microcentrifuge and denatured by heating for 5 minutes in boiling water bath and subsequent cooling in ice immediately for 5 minutes. It was spinned down, and 10µl reaction buffer added, mixed thoroughly, the 2 µl of labeling reagent added and mixed well but gently. 10 µl cross linker solution was added and briefly spinned down. The reaction was incubated for 30 minutes at 37 degrees C.
Post hybridization stringency washes were prepared for detection. A little primary buffer was put into a box and membrane placed into box, for washing. For the secondary wash, the second prepared buffer was added and membrane washed with gentle agitation. It was ensured that the membrane was maintained in wet condition.
A wrap film for placing the membrane was prepared, and membrane placed with the marked side up. 1.5 ml of CDP-star reagent was added on membrane and left to stand for more than 5 minutes, ready for detection under chemiluminescence.