Agarose gel electrophoresis experimental technique

related topicsAbstract:
agar
gel electrophoresis is a common experimental
for
nucleic acid analysis. Although many reference books have the introduction of this method El', but the actual operation of some techniques can not be obtained from the book, this paper introduces the experimental techniques of r agarose gel electrophoresis, these experiences are derived from many years of graduate teaching practice, for the first involved in
mulecological biology
experimenters have a certain guiding significance.
key words: agarose gel; electrophoresis;Practical Preventive Medicine August 13, 2006 Issue 4Practical Preventive, Aug. In 2006, Vol 13, No 4nucleic acid molecules are two-sex dissosis molecules, in the electrophoretic buffer above its isoelectric point, its base is not dissolved, and the phosphoric acid group is completely dissophed, nucleic acid molecules are negatively charged, electrophoresis migration to the positive pole. Agarose is mainly extracted from marine
forage plant
agar and modified by glycosylation, for a polymer chain linear molecule, the use of agarose gel as an electrophoresis support medium, play the function of molecular sieve, so that the size and composition of different nucleic acid molecules migration rate appears to be large differences, so as to achieve the purpose of separation. Agarose gel electrophoresis operation is simple and fast, by adjusting its use concentration, so that the resolution meets the requirements of most experiments, so it has become a common method of separating, identifying and purifying nucleic acid molecules. However, there are still many problems to pay attention to in the course of operation.1 Gel Production
1. 1 Gel concentration The concentration of the preparation gel changes according to the experimental needs, generally at 0. 8% to 2. Between 0%, if a preparation gel 100 ml, the gel can melt again, but with the increase in the number of melting, water loss is also more and more, the gel concentration will be higher and higher, resulting in unstable experimental results, rehydration method: First, the container marked before the gel scale, after cooking glue to add the corresponding moisture to the original scale; A rough approach is to obtain an empirical rehydration value by repeatedly under more constant cooking conditions. To ensure that the gel concentration is basically maintained at the original concentration. The nucleic acid staining agent ethium bromide can be added to the melted agarose with a final concentration of 0. 5 t x g/ml;1, 2 comb plate selection Generally each mold is equipped with a number of different tooth type comb plate, comb width, the formation of a large sample L volume, for

DNA

fragment recovery experiments, etc. ; The choice of comb plate is mainly to see how much of the sample, in general, the sample amount of hours as far as possible to choose a thin comb plate glue, at this time the electrophoresis strip dense and clear, easy to analyze the results. In addition, every time the glue should pay attention to the distance between the comb and the base plate at least 1 mm, otherwise, when pulling the comb plate is prone to damage to the bottom layer of the gel L, resulting in sample leakage after the sample. Of course, the damage of dot-like palate L is also related to the time and method of pulling the comb plate, the general gel needs to be cooled more than 30 min to pull the comb plate, emergency situation can be molded gel block in the 4 degrees C refrigerator cooling about 15 min, the method of pulling the comb plate is to place the gel tank in the electrophoresis tank electrophoresis buffer, and then vertically upward slowly force, because there is liquid lubrication, the comb plate is easy to pull out and damage the point L.
2 sample
samples need to be added to the sample buffer, because the sample buffer is added to the density of glycelor or sucrose, so that the sample sinks into the bottom of the L; The sample buffer storage is generally 6 × (10×), indicating that the concentration is six times the working concentration. When used, the sample buffer should be diluted to double the concentration. The sample method is to put the
piptor
basic vertical point sample L, with the other hand to help secure the lower end of the piper, piper gun head (Tip) tip into the sample sample sample L can be injected into the sample L, do not insert tip tip to the bottom L, and point on the appropriate DNA molecular weight standard, so-called suitable means that the sample DNA molecular weight size should be basically within the dna measurement standard range.
3 electrophoresis
connects the positive pole of the electrophoresis instrument to the positive pole of the electrophoresis tank, the negative pole is connected to the negative pole, and the nucleic acid is negatively charged, moving from the negative to the positive. The electrophoresis buffer in the electrophoresis tank and the electrophoresis buffer should be the same as the electrophoresis buffer, the electrophoresis buffer just did not have the gel 1 mm as well, the electrophoresis buffer too much is the current increased, the gel heat. The voltage added to the gel during electrophoresis generally does not exceed 5 v/cm (refers to the distance between positive and negative electrodes, not the length of the gel), electrophoresis time is generally 3O to 60 min, according to the experimental needs can also be adjusted appropriately, voltage increase, electrophoresis time shortened, nucleic acid strip is relatively not neat, not clear enough; In addition, if the sample swims very slowly or does not swim after electrophoresis, check that the seal strips at both ends of the mold have been removed.4 Results Analysis
The more successful electrophoresis result is that the molecular weight standard strip is neat and clear, the sample strip is also neat and clear, if the strip is blurred and dim, from the point of view of agarose gel electrophoresis alone, the possible reason: what is the quality and quantity of ethyl bromide ingots? Ethyl bromide ingots can be easily decomposed, the mother liquid preparation time is too long or improperly preserved (generally 4 degrees C light-avoiding preservation for one year effective), or the final concentration did not reach 0. 5 vg/ml; In particular, TAE buffer, generally used 2 to 3 times to replace, TBE buffer can be used about 10 times.
In practice, IT molecular weight standard small fragments are often found to be blurred, that is enough because the concentration of agarose gel generally does not exceed 20%, smaller nucleic acid fragments in its range of resolution, and EB with positive charge, electrophoresis will move towards negative pride, if the gel contains EB (0. 30 min in aqueous solution of 5 sg/m1, smaller fragments can be re-dyed: in addition, ethyl bromide (EB) is a medium-strength mutagent agent, the operation of
gloves
, and will be added to the EB dyeing liquid for marking, properly preserved.