Nucleic acid molecules are amphoteric dissociation molecules. In the electrophoresis buffer above its isoelectric point, their bases are inseparable, and all phosphate groups are dissociated, so the nucleic acid molecules are negatively charged and migrate to the positive electrode during electrophoresis. Agarose is mainly extracted from marine plant agar and modified by glycosylation, which is a linear molecule with polymer chains. As a supporting medium for electrophoresis, agarose gel plays the role of molecular sieve, which makes the fluidity of nucleic acid molecules with different sizes and conformations very different, thus achieving the purpose of separation. Agarose gel electrophoresis is simple and rapid. By adjusting its concentration, the resolution can meet the requirements of most experiments, so it has become a common method to separate, identify and purify nucleic acid molecules. However, there are still many problems to pay attention to in the operation process.

1 gel making

The gel concentration of 1. 1 varies according to the experimental needs, and is generally between 0.8% and 2.0%. If 100 ml gel is prepared at one time, the unused gel can be melted again, but with the increase of melting times, the more water is lost, the higher the gel concentration will be, resulting in unstable experimental results. The first method is to replenish water to the container. The second is to weigh before boiling the glue, and add water to the original weight after boiling the glue. The rough method is to get an empirical hydration value through constant boiling conditions for many times. So as to ensure that the gel concentration is basically maintained at the original concentration. The nucleic acid stain ethidium bromide can be added to the melted agarose, and the final concentration is 0.5t * g/ml；; It can also be dyed after electrophoresis.

Selection of comb plates for 1 and 2. Generally, each glue-making mold is equipped with a plurality of comb plates with different teeth, with wide teeth and large sample size, which are used for DNA fragment recovery experiments. On the contrary, the comb teeth are narrow and thin, and the sample volume is small, which is used to identify PCR products and enzyme digestion products. The selection of comb plate mainly depends on the sample size. Generally speaking, when the sample size is small, try to choose a thin comb plate to make glue. At this time, the electrophoresis bands are dense and clear, which is convenient for the result analysis. In addition, attention should be paid to the distance between the comb teeth and the bottom plate at least 1 mm every time the glue is made, otherwise the gel bottom layer will be easily damaged when the comb plate is pulled out, which will lead to the sample leakage after sampling. Of course, the damage of the sample L is also related to the drawing time and method of the comb plate. Generally speaking, the gel needs to be cooled for more than 30 minutes, and then the comb plate is pulled. In case of emergency, the molded gel block can be cooled in the refrigerator at 4℃ for about 65438±05min. The method of pulling out the comb plate is to put the glue-making tank into the electrophoresis buffer in the electrophoresis tank, and then slowly apply force vertically. Because of the lubrication of liquid, the comb plate is easy to pull out and it is not easy to damage the sample.

Two point sample

Sample buffer should be added when sampling, because glycerol or sucrose is added to the sample buffer to increase the density and make the sample sink to the bottom of L; In order to indicate the migration process of samples, two indicators, bromophenol blue and xylene blue, are generally added to the loading buffer (it is worth noting that the indicator is not a stain, and the DNA stain is ethidium bromide, and orange fluorescence can only be seen under the excitation of ultraviolet light). Generally, the storage solution of loading buffer is 6× (10×), which means its concentration is 6 times of the working concentration. When in use, the loading buffer should be diluted to twice the concentration. The sampling method is to vertically sample the pipette and fix the lower end of the pipette with the other hand. When the tip of the pipette enters the sample L, the sample can be injected into the sample L.. Never insert the tip into the bottom of the sample L and point to the appropriate DNA molecular weight standard. Suitably, the molecular weight of sample DNA should be basically within the range of DNA molecular weight standard.

3 electrophoresis

Connect the anode of the electrophoresis apparatus and the anode of the electrophoresis tank, and connect the cathode and the cathode. The nucleic acid is negatively charged and moves from the negative electrode to the positive electrode. The electrophoresis buffer in the electrophoresis tank should be the same as that used for gel, and it is best that the electrophoresis buffer is just less than gel1mm. If there is too much electrophoresis buffer, the current will increase and the gel will heat up. During electrophoresis, the voltage applied to the gel is generally less than 5 V/cm (referring to the distance between the positive and negative electrodes, not the length of the gel), and the electrophoresis time is generally 3o ~ 60min, which can be appropriately adjusted according to the experimental needs. With the increase of voltage, the electrophoresis time is shortened and the nucleic acid bands are relatively irregular and unclear. On the contrary, the voltage decreases, the electrophoresis time is longer and the nucleic acid bands are neat and clear. In addition, if the sample swims slowly or does not swim after electrophoresis, please check whether the sealing strips at both ends of the plastic mold have been removed.

4 Result analysis

The successful electrophoresis results show that the molecular weight standard band is neat and clear, and the sample band is neat and clear. If the band is fuzzy and dim, the possible reason is: from the perspective of agarose gel electrophoresis, what is the quality and quantity of ethidium bromide? Ethyl bromide is easy to decompose when exposed to light, and the mother liquor is prepared for a long time or stored improperly (usually effective within one year at 4℃ in the dark), or the final concentration does not reach 0.5 VG/ml; The buffer in the electrophoresis tank is used too many times, and the buffering capacity decreases. In particular, TAE buffer should be replaced after 2 ~ 3 times of use, while TBE buffer can be used about 10 times.

In practical work, it is often found that the standard small fragments of DNA molecular weight are vague, because the concentration of agarose gel is generally less than 20%, the smaller nucleic acid fragments are within its resolution range, and EB is positively charged, so it will move to negative potential during electrophoresis. If the gel is placed in an aqueous solution containing EB(0.5g/m 1) for 30 minutes, the smaller fragments can be stained again. In addition, ethidium bromide (EB) (0.5 # g/m 1).