Agarose electrophoresis
(1) Take a certain amount of the DNA sample to be tested and digest it with an appropriate restriction enzyme. The amount of DNA depends on the type of sample and
The purpose of the experiment varies, and for the restriction endonuclease map analysis of the cloned fragment, 0.1-0.5 μg can be taken;
Single-copy gene sequence in genomic DNA requires 10-20 μg; when using oligonucleotide probe or probe specific emission
When the sexual activity is low, 30 to 50 μg is required.
(2) The enzyme was cut and electrophoresed on an agarose gel.
(3) After the end of electrophoresis, EB staining was performed, and the electrophoresis results and photographs were observed under long-wave ultraviolet rays.
2. Imprint transfer
(1) Cut the gel and mark it (removed in the lower left corner) to facilitate positioning, and then place the gel in a container.
(2) Soak the gel in an appropriate amount of denaturing solution, let it denature at room temperature for 1 h, and gently shake it without interruption.
(3) The gel was rinsed once with deionized water, then immersed in an appropriate amount of neutralizing solution for 30 min, and gently shaken without interruption. Replace the neutralizing solution and continue to soak for 15 min.
(4) The filter paper and the NC film were cut into the same size as the glue, and the NC film was immersed in the transfer buffer for 30 min.
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(5) Operation according to the figure: flatten the layers layer by layer, and leave no bubbles or wrinkles between the layers.
(6) Siphon transfer for 12-16h.
3. Fix DNA
(1) The transferred NC membrane was taken out, and the NC membrane was blotted with a filter paper, and the NC membrane was lightly flattened upward in a denaturant for 5 min.
(2) The NC film was spread on a neutral solution in the same manner, and neutralized twice, each time for 5 minutes.
(3) The film was sandwiched between two dry filter papers and baked at 80 ° C for 1-2 h.
Prehybridization
(1) The film was immersed in 5 x SSC for 2 min, and the film was placed in a clean plastic bag.
(2) The pre-hybrid solution was preheated at 65 ° C in advance, and then 10 ml of the pre-hybrid solution was added to the bag and sealed.
(3) Pre-hybridization at 65 ° C for more than 1 h, shaking from time to time.
Hybrid
(1) Take out the hybridization bag, cut off a corner to remove the hybridization solution, add 5 ml of the hybridization solution and 5 μl of the denatured probe to remove the bubbles and seal.
(2) The hybridization bag was mixed in a 65 ° C water bath overnight.
6. Wash the film under the following conditions
2×SSC, 0.1% SDS 50ml room temperature 5min /2 times
0.1×SSC, 0.1% SDS 50ml 65°C 15min /2 times
7. Immunological enzyme detection
(1) Coupling reaction
1 Wash the membrane with neutralizing solution for 2 min at room temperature.
2 Wash the membrane with 50 ml of blocking solution for 30 min, shake at room temperature.
3 Dilute the antibody-Dig-Ap to 750 mU/ml with a neutralizing solution, seal the membrane into the hybridization bag, and gently incubate for 5 min by adding 5 ml of the diluted antibody.
4 Wash the membrane with 50 ml of neutralizing solution, 10 min × 2 times, shake at room temperature, and remove unbound antibody.
(2) Color reaction 1 Balance the membrane with an equilibration solution of 20 ml for 2 min.
2 Put the membrane into the hybrid bag, add 5ml of coloring solution, avoid the light for about 30min, and do not shake when the color appears.
3 Wash the membrane with TE to terminate the reaction.
Bake at 480 ° C.
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