1 Gel production
1.1 Gel concentration The concentration of the prepared gel according to the experimental needs of the change, generally in the 0.8% ~ 2.0%, if a preparation of gel 100 ml, not used up the gel can be melted again, but with the increase in the number of times of melting, the loss of water is also more and more water, the concentration of gel is higher and higher, resulting in the results of the experiments are unstable, replenishment of water methods: First, mark on the container before cooking the gel, the water loss is also more and more. Mark the scale on the container before cooking the gel, and replenish the corresponding water to the original scale after cooking the gel; the second is to weigh the gel before cooking, and replenish the water to the original weight after cooking the gel. Roughly speaking, the method is to obtain an empirical water replenishment value through several times of more constant cooking conditions. To ensure that the gel concentration is basically maintained at the original concentration. The nucleic acid stain ethidium bromide can be added to melted agarose at a final concentration of 0.5 t*g/ml; it can also be stained at the end of electrophoresis.
1, 2 comb plate selection Generally, each glue mold are equipped with a number of different teeth comb plate, comb teeth wide and thick, the formation of a larger volume of point sample solenoid, used for DNA fragmentation recovery experiments, etc.; on the other hand, the comb teeth are narrow and thin, the formation of the point sample solenoid volume is small, used for PCR products, enzyme digestion product identification, etc.. The choice of comb plate is mainly depends on the amount of sample, in general, the amount of sample as small as possible to choose a thin comb plate gel, this time the electrophoresis band is dense and clear, easy to analyze the results. In addition, each time the gel should pay attention to the distance between the comb teeth and the bottom plate should be at least 1 mm, otherwise, the bottom layer of the gel solenoid will be easily damaged when pulling out the comb plate, which will lead to the leakage of the sample after the sample is dispensed. Of course, the destruction of the spotting solenoid is also related to the time and method of pulling the comb plate, generally the gel needs to be cooled for more than 30 min before pulling the comb plate, in case of emergency, you can put the molding of the gel block in the refrigerator at 4 ℃ to cool down for about 15 min, the method of pulling the comb plate is to place the gel tank in the electrophoresis buffer in the electrophoresis tank, and then vertical upward and slowly exert force, because of the lubrication of the liquid, the comb plate is easy to pull and not easy to damage the spotting solenoid.
2 Spotting
Spotting needs to be added to the sample buffer, because of the addition of glycerol or sucrose to increase the density of the sample buffer, so that the sample sinks to the bottom of the solenoid; indication of the migration process of the sample, the sampling buffer is generally added to two indicators, bromophenolan and xylene cyanide (it is worth noting that the indicator is not a stain, DNA stain is ethidium bromide, and to be The DNA stain is ethidium bromide and is only visible as orange fluorescence under UV excitation). Sampling buffer stocks are typically 6× (10×), indicating that they are six times the working concentration. Sampling buffer should be diluted to double the concentration at the time of use. Sampling method is the pipette is basically perpendicular to the point of sampling solenoid, with the other hand to help fix the lower end of the pipette, pipette tip (Tip) tip into the point of sampling solenoid can be injected into the solenoid samples, do not Tip inserted to the bottom of the solenoid, and point on the suitable DNA molecular weight standard, the so-called suitable means that the sample DNA molecular weight size should be basically within the DNA molecular weight standard.
3 Electrophoresis
Connect the positive pole of the electrophoresis instrument with the positive pole of the electrophoresis tank, and the negative pole with the negative pole, the nucleic acid is negatively charged, and moves from the negative pole to the positive pole. The electrophoresis buffer in the electrophoresis tank should be the same as the electrophoresis buffer used for making the gel, and the electrophoresis buffer should be just 1 mm above the gel, and the electrophoresis buffer should be too much for the current to increase and the gel to be heated. Electrophoresis on the gel voltage generally does not exceed 5 v / cm (refers to the distance between the positive and negative electrodes, rather than the length of the gel), electrophoresis time is generally 3O ~ 60 min, according to the needs of the experiment can be adjusted appropriately, the voltage increases, electrophoresis time is shortened, the nucleic acid bands are not neat and clear; on the other hand, the voltage decreases, the electrophoresis time is longer, the nucleic acid bands are neat and clear. On the other hand, if the sample moves slowly or does not move after electrophoresis, please check whether the sealing tape at both ends of the mold has been removed.
4 Result analysis
The more successful electrophoresis result is the molecular weight standard band is neat and clear, and the sample band is also neat and clear, if the band is vague and dark, from the perspective of agarose gel electrophoresis alone, the possible reasons: what is the quality and quantity of ethidium bromide? Ethidium bromide is easy to decompose in the presence of light, the preparation time of mother liquor is too long or improperly preserved (generally 4 ℃ light preservation is effective for one year), or the final concentration is not up to 0.5 vg / ml; buffer in the electrophoresis tank is used too often, the buffer capacity decreases. Especially TAE buffer, generally used 2 to 3 times to be replaced, TBE buffer can be used about 10 times.
In practice, often found in the DNA molecular weight standard small fragments of fuzzy, that's because the agarose gel concentration is generally not more than 20%, the smaller nucleic acid fragments within its resolution, and EB positively charged, electrophoresis will be moved to the negative Pride, if the gel is placed in an aqueous solution containing EB (0.5#g/m1) for 30 minutes, the smaller fragment can be re-stained. In addition, ethidium bromide (EB) is a medium-strength mutagen, so gloves should be worn during the operation, and the staining solution with EB should be labeled and stored properly
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