2. After mixing the reaction system, place the eppendorf tube on a proper support (such as inserting it on a foam plastic board), and keep the temperature in a water bath at 37℃ for 2-3 hours to complete the enzyme digestion reaction.
3. Add 2μl 0. 1mol/L EDTA(pH8.0) to each tube, mix well to stop the reaction, and store in the refrigerator for later use. 1, take 20ml of 5×TBE buffer and add water to 200ml to prepare 0.5×TBE dilution buffer for later use.
2. Preparation of glue solution: Weigh 0.4g of agarose, put it in a 200ml conical flask, add 50ml 0.5×TBE dilution buffer, put it in a microwave oven (or electric stove) and heat it until the agarose is completely melted, then take it out and shake it well, which is 0.8% agarose gel solution. Shake it from time to time during heating, so that agarose particles attached to the bottle wall enter the solution. When heating, the sealing film should be covered to reduce water evaporation.
3. Preparation of rubber sheet: The two ends of the plexiglass rubber tank are sealed tightly with rubber plaster (width about 1cm). Place the sealed rubber groove on the horizontal support, insert the sample comb, and observe that the lower edge of the comb teeth should keep a gap of about 1mm from the bottom of the rubber groove.
Add the solution of ethidium bromide (EB) to the agarose gel cooled to 50-60℃ to make its final concentration be 0.5μg /ml (EB may not be added to the gel, but soaked and dyed with 0.5μg/ml EB solution after electrophoresis). Use a pipette to absorb a small amount of melted agarose gel to seal the inside of the adhesive plaster. After the agarose solution solidifies, carefully pour the remaining agarose into the adhesive tank to form a uniform adhesive layer. The temperature when pouring the glue should not be too low, otherwise the solidification will be uneven and the speed should not be too fast, otherwise bubbles will easily appear. Pull out the comb after the glue is completely solidified, taking care not to damage the gel at the bottom of the comb, and then add 0.5×TBE dilution buffer into the tank until the liquid level just passes the upper surface of the glue plate. Because of the edge effect, there will be some bumps near the sample tank, which will prevent the buffer from entering the sample tank, so it is necessary to ensure that the sample tank should be filled with buffer.
4. Sample addition: Take 10μl enzymolysis solution and 2μl 6× sample carrier solution, and carefully add them into the sample tank with a micro pipette. If the DNA content is low, the sample loading can be increased according to the above ratio, but the total volume should not exceed the capacity of the sample tank. Tip head should be replaced after each sample is added, so as to prevent mutual contamination. Be careful when loading samples, so as to avoid damaging the gel or piercing the gel at the bottom of the sample tank.
5. Electrophoresis: After adding the sample, close the cover of the electrophoresis tank and turn on the power immediately. The control voltage is kept at 60-80V, and the current is above 40mA. When the bromophenol blue band moves to about 2cm from the gel front, electrophoresis is stopped.
6. Dyeing: After electrophoresis, the rubber plate without EB is moved into 0.5μg/ml EB solution and dyed for 20-25 minutes at room temperature.
7. Observation and photographing: Observe the stained or EB-added electrophoretic rubber sheet under a long-wavelength ultraviolet lamp with a wavelength of 254nm. The presence of DNA shows orange-red fluorescent bands that can be distinguished by naked eyes. Wear protective glasses or plexiglass mask when observing under purple light to avoid eye damage. After the camera lens is added with a close-up lens and a red filter, the camera is fixed on the photo frame, using panchromatic film, with an aperture of 5.6 and an exposure time of 10- 120 seconds (selected according to the depth of fluorescent strips).
8. Preparation of DNA molecular weight standard curve: Based on the amplified electrophoresis photos, the migration distance of EcoRⅠ ⅰ and HindⅢ ⅲ fragments of λDNA was measured with calipers, in centimeters. Taking the common logarithm of nucleotide number as the ordinate and migration distance as the abscissa, a smooth curve connecting all points is drawn on the coordinate paper, which is the standard curve of DNA molecular weight under this electrophoresis condition.
9. Determination of DNA fragment size: Measure the migration distance of each fragment of the DNA sample with calipers on the amplified electrophoresis photos, and according to this value, find out the corresponding logarithmic value on the standard curve of DNA molecular weight, and further calculate the molecular weight of each fragment (if the standard curve is drawn with single logarithm graph paper, the size of DNA fragment can be directly found out according to the migration distance). On the other hand, if the size of DNA fragment is known, its expected migration distance can also be found out from the standard curve.
10, Determination of the sequence of DNA restriction fragments: According to the electrophoresis analysis results of single restriction, double restriction and multi-restriction, logical reasoning is carried out according to the data of the size of DNA restriction fragments, and then the sequence of each restriction fragment and the relative position of each restriction site are determined. The restriction endonuclease map of the DNA molecule can be obtained by ring diagram or straight line diagram.
[attention]
1. The volume of the added DNA solution should not be too large, otherwise other components in the DNA solution will interfere with the enzyme reaction.
2. The enzyme activity is usually expressed by the enzyme unit (U). The definition of the enzyme unit is: under the optimum reaction conditions, the enzyme amount of 1 hour is one unit, but the DNA prepared in many experiments is not as easy to degrade as lDNA, so it is necessary to increase the amount of enzyme. It is not appropriate to add too much enzyme to the reaction solution. Besides considering the cost, trace impurities in the enzyme solution may interfere with the subsequent reaction.
3. Generally, the enzyme sold in the market has a large concentration. For the sake of economy, it can be diluted with enzyme reaction buffer (1×) in advance. In addition, the enzyme is usually stored in 50% glycerol. In the experiment, the glycerol concentration in the reaction solution should be controlled below110, otherwise, the enzyme activity will be affected.
4. The observation of DNA is inseparable from the ultraviolet transmission instrument, but ultraviolet light can cut DNA molecules. When recovering DNA from the glue, we should shorten the illumination time as much as possible and use a long-wavelength ultraviolet lamp (300-360nm) to reduce the cutting of DNA by ultraviolet light.
5. EB is a strong mutagen with moderate toxicity. Wear gloves when preparing and using it, and don't spill EB on the desktop or ground. All containers or articles contaminated with EB must be specially treated before being cleaned or discarded.
6. When there is too much EB, the glue is dyed too deeply and the DNA band is unclear, you can put the glue in distilled water and observe it after 30 minutes.