DNA's agarose gel electrophoresis.

related topics

.I. PrincipleTHE
is negatively charged in an alkaline solution and therefore moves towards the positive pole under the action of an electric field. In
agar
sugar gel electrophoresis, because agarose gel has a certain aperture, different lengths of DNA molecules due to the gel's deterrent action size, the speed of migration is different, so that according to the molecular weight size can be effectively separated. Ethyl bromide can be inserted into the double strands of DNA molecules. Under ultraviolet light, the DNA inserted into the ethyl bromide ingot is orange-red fluorescent, so ethyl bromide can be used as a fluorescent indicator to indicate the DNA content and location., materials,
equipment
and
reagents
(i) materials:
molecules
different amounts of DNA fragments.
(ii) instruments and equipment: 1. electrophoresis; 2. UV detector; 3. horizontal electrophoresis tank; 4.
. Liquidator
;5. Disposable
gloves
.
(iii) Reagents: 1. pH8.3 Tris-boric acid-
. EDTA
buffer: 10.78g Tris, 5.500g boric acid, 0.930g EDTA-Na2 dissolved in ionic water, fixed to 100ml, diluted 10 times in time; EB solution: ethyl bromide ingots (10mg/ml) (diluted 10 times over time); Sample buffer: 50% glycelin plus 0.25% bromophenol blue;3, experimental steps
(i) preparation of agarose gel: 1. Said to take agar sugar 1g, add 10 times electrophoresis buffer 10 ml, then add distilled water 90 ml, in the electric furnace
heating
dissolved, with 1% agarose gel; 2. Seal both ends of the electrophoresis template, pour in the agarose gel solution and insert the comb. 3. After condensation, set out the comb and place the electrophoresis gel in the electrophoresis tank.
(ii) sample: take the sample solution 20 μl, add 1/5 volume plus sample buffer, mix well, add the solution to the sample hole, and add standard molecular weight DNA to another sample hole. In general, DNA samples are best controlled between 0.5 and 1.0 μg.
(iii) electrophoresis: add pH8.3Tris-boric acid-EDTA buffer, power to maintain 2 to 4V/cm, electrophoresis to bromphenol blue to the bottom edge of the glue to stop electrophoresis.
(iv) staining and observation: electrophoresis finished, take out the gel mold, push it to a clean glass plate, in 254nm or 300nm wavelength UV lamp observation, DNA presence position present orange-yellow fluorescence, placement time of more than 4 to 6h fluorescence weakened, so should immediately use domestic full-color film to take pictures, and should be added red filter.
.