Classification of nucleic acid electrophoresis

1. agarose is a linear polymer extracted from seaweed. Agarose is heated and melted in the required buffer to form a clear and transparent sol, and then poured into a rubber mold. After curing, a solid matrix will be formed, and its density depends on the concentration of agarose.

When the gel is placed in an electric field, the charged nucleic acid migrates to the anode through the grid of the gel at neutral pH, and the migration rate is affected by molecular size, conformation, agarose concentration, applied voltage, electric field, electrophoresis buffer and embedded dye amount. After electrophoresis for a proper time under different conditions, nucleic acid fragments with different sizes and conformations will be in different positions on the gel, thus achieving the purpose of separation. The separation range of agarose gel is wide, and DNA with a length of 200 BP-50kb can be separated by agarose gel with various concentrations.

2. Instruments and reagents for agarose gel electrophoresis should include horizontal gel electrophoresis tank and its supporting electrophoresis comb, voltage-stabilized electrophoresis instrument, microwave oven or ordinary electric furnace. At the same time, it is equipped with ultraviolet detector and camera system.

The reagents include agarose, electrophoresis buffer, ethidium bromide solution and gel loading buffer.

Electrophoresis buffer is usually TBE( 1 000ml contains 5.4g Tris, 2.75g boric acid, 2ml 0.5 mol/L EDTA, pH8.0).

Ethidium bromide (EB) is a fluorescent dye, which can be embedded between pairs of bases of double-stranded nucleic acids and migrate with nucleic acid fragments during electrophoresis. When the gel is placed under ultraviolet light, EB inserted into the nucleic acid chain generates red fluorescence under the excitation of ultraviolet light, which can clearly show the migration of each nucleic acid fragment. EB is easily decomposed by light and should be stored in brown reagent bottle at 4℃. As EB is a strong mutagen with moderate toxicity, it must be operated with gloves.

There are four commonly used gel loading buffers, as shown in the following table: gel loading buffer buffer type 6× buffer formula storage temperature ⅰ0.25% bromophenol blue.

0.25% xylene blue

40%(W/V) sucrose aqueous solution 4℃ Ⅱ 0.25% bromophenol blue

0.25% xylene blue

15%(W/V) sucrose aqueous solution at room temperature Ⅲ 0.25% bromophenol blue

0.25% xylene blue

30% (w/v) glycerol solution at 4℃ ⅳ 40% (w/v) sucrose solution at 4℃ 3. Preparation and electrophoresis of gel.

The operation method is as follows:

(1) Seal the edge of the glass plate or plastic disk with the electrophoresis device with transparent glue, make a glue mold, and put it on the horizontal workbench;

(2) Weighing an appropriate amount of agarose, placing it in electrophoresis buffer, and heating to dissolve the agarose;

(3) When the solution is cooled to 60℃, add 10mg/ml EB storage solution to make the final concentration reach 0.5 mg/ml;

(4) Place the electrophoresis comb 0.5- 1mm away from the bottom plate of the plastic mold, and pour the agarose solution into the plastic mold with a thickness of about 3-5mm, taking care to avoid bubbles;

(5) After the gel is completely cured, remove the comb and transparent glue, and put the gel into the electrophoresis tank. Add TBE buffer to make it just pass through the bonding surface about1mm;

(6) mixing the DNA sample with 1/6 sample adding buffer, and adding it into the sample tank;

(7) Turn on the power supply, make the sample tank at the negative end, and use the voltage of 1-5v/cm for electrophoresis for a proper time;

(8) After electrophoresis, the gel containing EB can be directly placed on an ultraviolet detector for observation and photo recording, or the gel without EB can be dyed in 0.5μg/ml EB solution for 30-45 minutes, and then the results can be recorded by observing and photo recording as described above.

4. Gel photography

Need to configure 135 camera, panchromatic 135 film, camera bracket, close-up lens and red filter, plexiglass protective mask.

The operation needs to be performed in a dark room. Fix the camera, put the gel in the proper position on the ultraviolet detector, focus, install the red filter and take pictures as usual.

Automatic gel treatment system can also be used, but the instrument cost is high.

5. purification 5. EB solution

Because EB has certain toxicity, after the experiment, the solution containing EB should be purified and then discarded to avoid polluting the environment and endangering human health.

The solution with (1)EB content greater than 0.5μg/ml can be treated as follows:

(1) dilute EB solution with water to a concentration below 0.5 μ g/ml;

(2) adding 1 KMnO4 with a volume of 0.5mol/L, uniformly mixing, adding HCl with an equal volume of 25mol/L, uniformly mixing, and standing at room temperature for several hours;

③ Add 1 2.5mol/L NaOH, mix well and discard.

(2) The solution with 2)EB content less than 0.5μg/ml can be treated as follows:

(1) according to the amount of 1mg/ml, add activated carbon, gently shake it from time to time, and leave it at room temperature 1 hour;

② Filter with filter paper, seal the activated carbon with filter paper and discard it. 1. Principle Polyacrylamide gel is formed by chemical reaction of acrylamide monomer, chain polymerization catalyst N, N, N', N'- tetramethylethylenediamine (TEMED), ammonium persulfate and crosslinking agent N, N'- methylene bisacrylamide. Acrylamide monomer is polymerized under the action of catalyst to form long chain, which is crosslinked by crosslinking agent to form gel, and its pore size is determined by chain length and crosslinking degree. The chain length depends on the concentration of acrylamide, and the crosslinking degree of the polymer can be changed by adjusting the concentration ratio of acrylamide to crosslinking agent.

Polyacrylamide gel electrophoresis can achieve the purpose of separation according to the charge, molecular size and shape of electrophoretic samples, which has both molecular sieve and electrostatic effect and has higher resolution than agarose gel electrophoresis. DNA fragments with a difference of only 1 nucleotide can be isolated.

Polyacrylamide gel electrophoresis is used to analyze and prepare DNA fragments with the length less than 1kb. According to the size of the nucleic acid fragment to be separated, gels with different concentrations can be prepared.

2. Gel preparation and electrophoresis Because oxygen can inhibit the polymerization of acrylamide, the filling of polyacrylamide gel is often carried out between the interlayer formed by two closed glass plates. In this device, only the gel on the top layer is in contact with oxygen in the air, thus greatly reducing the inhibition of oxygen on polymerization. Polyacrylamide gel electrophoresis generally adopts vertical device.

Gel preparation and electrophoresis operations are as follows:

(1) preparation reagent

① 30% acrylamide: 100ml double distilled water contains 29g acrylamide and 1g N, N'- methylene bisacrylamide.

② Every liter of 5× TBE solution contains 54g Tris. Hydrochloric acid, 27.5g of boric acid and 20ml of 0.5mol/L EDTA(pH8.0).

③ 10% ammonium persulfate: 10ml double distilled water contains 1g ammonium persulfate.

(2) Scrubbing the glass plate and gasket strips with detergent in the rubber mold of the device in advance, rinsing with tap water and deionized water, and drying. During installation, put the larger glass plate flat on the workbench, put two pads on both sides of the glass plate, and apply a small amount of vaseline. Put the upper glass plate on the pad, clamp the glass plate and the pad with a clamp, and seal the bottom with 1% agarose. In order to prevent glue leakage, all three sides of the comb should be sealed with waterproof tape except one side.

(3) Calculate the required amount of gel solution according to the glass plate size and interlayer thickness, and prepare the solution (100ml) according to the following table. The concentration of polyacrylamide gel solution is 3.5 5.0 8.0 12.0 20.0 30% acrylamide (ml) 1 16.626.40.066 water (ml). 67.7 62.7 52.7 39.312.7 5× tbe (ml) 20.0 20.0/0% ammonium persulfate (ml)0.7 0.7 0.7 0.7 0.7 0.7 0.7 Add 35μl TEMED to 100ml mixed solution and mix.

(4) Insert the electrophoresis comb immediately to prevent bubbles from forming under the comb teeth.

(5) After polymerization for 65438 0 hours at room temperature, refractive bands appeared under the comb teeth, indicating that the polymerization reaction was completed. If the gel is not used immediately, it can be covered with gauze or filter paper (soaked in 1×TBE) and stored at 4℃ for 1-2 days.

(6) Pull out the comb, and immediately flush the sample adding hole with water.

(7) Remove the bottom adhesive tape and put the gel vertically into the electrophoresis tank. Fill the upper and lower tanks with 1×TBE solution to drive away bubbles attached to the bottom of the gel. Clean the injection hole with 1×TBE solution;

(8) Mix the nucleic acid sample with an appropriate amount of 6 times gel loading buffer (see Table 2-28) and add it to the gel loading hole;

(9) Turn on the power supply, and the anode is connected with the lower tank. The voltage is generally controlled at 1.8v/cm. When the voltage is too high, the heat generated by the gel will lead to the bending of DNA bands and even the melting of small DNA fragments.

(10) After electrophoresis, remove the glass plate and gel, put them on the workbench, pry them gently from the corner of the interlayer, gently remove the glass plate above, carefully take out the gel, dye them in the dyeing solution, and observe the results.

3. Gel staining and observing the staining of nucleic acid bands in polyacrylamide gel, ethidium bromide method and silver staining method are commonly used. The staining method of the former is the same as that of agarose gel.

The silver staining method has high sensitivity and the operation is as follows:

(1) Fix the gel in static solution (10% ethanol, 0.5% glacial acetic acid) 10 min;

(2) Double steaming and water washing 1-2 times;

(3) putting it into AgNO3 solution of 0.0 1mol/L, and reacting at room temperature for 1.5-30 minutes;

(4) full washing;

(5) put it into NaOH- formaldehyde mixed solution (200ml 3% NaOH, containing 1ml formaldehyde) to react until the band color is clear and the background is suitable;

(6) Terminate the reaction with 5% glacial acetic acid.