Tris (trihydroxymethylaminomethane) is a weak base with a pKa of 8.1 at room temperature of 25℃ and an effective buffer range of pH 7.0 ~ 9.2.The pH of aqueous solution of Tris alkali is about 10.5. Hydrochloric acid is generally added to adjust the pH value to the desired value, so that the buffer of this pH value can be obtained.Since Tris buffer is a weakly alkaline solution, DNA will be deprotonated in such a solution, thus improving its solubility.If the pH-adjusted acid solution is replaced with acetic acid, a "TAE buffer" (Tris/Acetate/EDTA) is obtained, while a "TBE buffer" (Tris/Borate/EDTA) is obtained by replacing it with boric acid.These two buffers are commonly used in nucleic acid electrophoresis experiments.TAE, TBE, etc. prepared by Tris are the most commonly used reagents for DNA electrophoresis, and TE (pH 8.0) is mainly used to dissolve DNA.(TE is a combination of Tris and EDTA.)1MTris-HCl6.8 and 1.5MTris-HCl8.8 are the most commonly used reagents for SDS-PAGE.
Among the conventional operations of genetic engineering, agarose gel electrophoresis is the most widely used.Agarose gel electrophoresis is a conventional method for separating, identifying and purifying DNA fragments.It usually uses a horizontal electrophoresis device to electrophoresis under an electric field with constant intensity and direction.DNA molecules are negatively charged in gel buffers (generally alkaline) and migrate from negative to positive electrodes in an electric field.The rate of DNA molecule migration is dependent on the size and conformation of the molecule.The influence of electric field strength and direction, base composition, temperature and embedded dyes, etc.
① TE buffer: (10 mmol/L Tris-HCl, 1 mmol/L EDTA)
Electrophoresis buffer (50XTAE)
② Electrophoresis buffer (50XTAE): Tris 242g, glacial acetic acid 57.1ml, EDTA (0.5mol/L pH 8.0) 100ml
Dilute 50 times with distilled water when used.
③ Sample buffer (6X): 0.25% bromophenol blue, 0.25% xylene blue, 40% (W/V) sucrose
④ STET buffer (pH 8.0) (8% sucrose, 0.5% Triton, 50 mmol/L EDTA, 10 mmol/L Tris)
1) Measure 100 ml of TAE electrophoresis solution, add 0.7 g of agarose, mix well, place in microwave oven, heat for 3 minutes, fully dissolve agarose.
2) Close the clean and dry electrophoretic plate with sterilized rubber and seal the edge with a small amount of agarose solution.Place the comb and adjust the distance between the bottom edge of the comb and the electrophoresis plate, generally 1-2 mm is appropriate.
3) When the dissolved agarose solution is cooled to about 50C, add 5 M L of ethidium bromide, and the final concentration of ethidium bromide is 1.0 m g/m L. After mixing, pour the agarose solution into the electrophoresis plate and keep it still without moving.
4) After the gel has completely solidified (30-45 minutes at room temperature), gently remove the comb, remove the tape, and place the gel in the electrophoresis pad.
5) In the electrophoresis process, electrophoresis solution (1'TAE) was added to cover the agarose gel surface with about 1-2 mm electrophoresis solution.
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