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How to prepare the commonly used electrophoresis buffers,

2019-05-17 来源:亚科官网
Electrophoresis buffer is an important component of nucleic acid and protein gel electrophoresis systems. It is a conductor in the electrophoresis field and a necessary condition for maintaining a constant pH of the electrophoresis system. It refers to the buffer solution used in molecular electrophoresis to stabilize the pH of the system. Common nucleic acid electrophoresis buffers are: TAE, TBE, TPE and MOPS.
The role of electrophoresis buffer
One of the roles of the buffer during electrophoresis is to maintain a suitable pH. Electrolysis reaction occurs between the anode and the cathode during electrophoresis. The oxidation reaction occurs in the anode (4OH--4e->2H2O+O2), and the reduction reaction occurs in the cathode (4H++4e->2H2). Long-term electrophoresis will cause The anode becomes sour and the cathode becomes alkali. A good buffer system should have a strong buffering capacity so that the pH of the two poles of the solution remains essentially the same.
Another function of the electrophoresis buffer is to make the solution have certain conductivity to facilitate the migration of DNA molecules. For example, the general electrophoresis buffer should contain 0.01-0.04 mol/L of Na+ ions, and the concentration of Na+ ions is too low. The speed is slow; too high will cause excessive current to cause the glue to heat up or even melt.
Another component of the running buffer is EDTA, which is added at a concentration of 1-2 mmol/L to sequester Mg2+ plasma to prevent activation of DNase during electrophoresis and to prevent precipitation of Mg2+ ions and nucleic acids.
Method for preparing electrophoresis buffer
MOPS Buffer, 3-morpholine propanesulfonic acid buffer, is a biological buffer that can be used as an electrolyte system component for two-dimensional gel electrophoresis (ECF); it can also be used for Northern hybridization as a separation of RNA and Buffer when transferring film.
The preparation method of commonly used 10×MOPS Buffer is as follows:
(1) Weigh 41.8 g of MOPS and dissolve it in about 700 mL of DEPC treated water;
(2) using 2N NaOH to adjust the pH to 7.0;
(3) adding DEPC-treated 1M NaOAC 20mL, 0.5 M EDTA (pH 8.0) 20 mL to the solution;
(4) Make up to 1 L, filter with 0.45 μm filter to remove impurities, and store at room temperature in the dark.
2. TAE
TAE is the most widely used buffer system. It is characterized in that the supercoil is more in line with the actual relative molecular mass (the relative molecular mass measured during electrophoresis in TBE is greater than the actual molecular mass), and the mobility of double-stranded linear DNA is better than the other two (TAE). It is about 10% faster than TBE) buffer. When electrophoresing a fragment larger than 13 kb, TAE buffer will achieve better separation. In addition, the TAE buffer system can be easily used for electrophoresis when recovering DNA fragments.
50×TAE Buffer preparation method:
1. Weigh 242g of Tris and 18.612g of EDTA in a 1L beaker;
2. Add about 800ml of deionized water to the beaker and mix well;
3. Add 57.1 ml of glacial acetic acid and dissolve completely;
4. Adjust the pH to 8.3 with NaOH, dilute to 1 L with deionized water, and store at room temperature.
Dilute 50 times or 100 times when used, ie 1 × TAE Buffer or 0.5 × TAE.
3. TBE
TBE (Tris boric acid) is a buffer used in PCR technology.
TBE formula:
1, the use of liquid (0.5 ×)
0.045 mol/L Tris-boric acid 0.001 mol/L EDTA was dissolved in sterile water and the volume of the solution was 1 L.
2. Concentrated stock solution (5×)
54g Tris base 27.5g boric acid 20ml 0.5mol/L EDTA Dissolve and mix in sterile water, adjust pH to 8.0 with NaOH, and solution volume 1L.
Related links: MOPS
Edited by Suzhou Yacoo Science Co., Ltd.