First, the characteristics of agarose gel
Natural agar (agar) is a polysaccharide consisting mainly of agarose (about 80%) and agaropectin. Agarose is a neutral substance composed of galactose and its derivatives, and has no charge. Agarose gel is a strongly acidic polysaccharide containing sulfate and carboxyl groups. Because these groups have a charge, they can be produced under the action of an electric field. Strong electroosmosis, combined with sulfate can affect the electrophoresis speed and separation effect with certain proteins. Therefore, at present, agarose is often used for electrophoresis support for plate electrophoresis, and the advantages thereof are as follows.
(1) Agarose gel electrophoresis is easy to operate, and the electrophoresis speed is fast. The sample can be electrophoresed without prior treatment.
(2) The agarose gel has a uniform structure and a large water content (about 98% to 99%). It is approximately free electrophoresis. The sample diffuses more freely and has little adsorption to the sample. Therefore, the electropherogram is clear, the resolution is high, and the repeatability is high. it is good.
(3) Agarose is transparent and UV-free, and the electrophoresis process and results can be directly detected and quantified by ultraviolet light.
(4) After electrophoresis, the zone is easy to stain, and the sample is easy to elute, which is convenient for quantitative determination. Made into a dry film can be stored for a long time.
At present, agarose is commonly used as an electrophoresis support to separate proteins and isozymes. Combining agarose electrophoresis with immunochemistry, it develops into an immunoelectrophoresis technology, which can identify complex systems that cannot be identified by other methods. Due to the establishment of ultra-micro technology, 0.1 ug of protein can be detected.
Agarose gel electrophoresis is also commonly used to isolate and identify nucleic acids, such as DNA identification, DNA restriction endonuclease mapping, and the like. Because of the convenient operation, the simple equipment, the small sample amount and the high resolution, the method has become one of the commonly used experimental methods in genetic engineering research.
Second, DNA agarose gel electrophoresis
The separation of nucleic acids by agarose gel electrophoresis is mainly based on their relative molecular mass and molecular configuration, and is also closely related to the concentration of the gel.
1. Relationship between nucleic acid molecule size and agarose concentration
(1) Size of DNA molecule In the gel, the migration distance (mobility) of the DNA fragment is inversely proportional to the logarithm of the base pair, so the distance moved by the standard of known size is compared with the moving distance of the unknown fragment. , the size of the unknown segment can be measured. However, when the size of the DNA molecules exceeds 20 kb, it is difficult to separate them by ordinary agarose gels. At this time, the mobility of the electrophoresis is no longer dependent on the molecular size, and therefore, when the DNA is separated by agarose gel electrophoresis, the molecular size should not exceed this value.
(2) The concentration of agarose is shown in the following table. Different sizes of DNA need to be separated by electrophoresis using different concentrations of agarose gel.
Table agarose concentration and DNA separation range
Agarose concentration /% 0.3 0.6 0.7 0.9 1.2 1.5 2.0
Linear DNA size / kb 60-5 20-1 10-0.8 7-0.5 6-0.4 4-0.2 3-0.1
2. Relationship between nucleic acid configuration and separation by agarose gel electrophoresis
The order of movement speed of DNA of different configurations is: covalently closed circular (cccDNA)>straight DNA>open-loop double-stranded circular DNA. When the agarose concentration is too high, the circular DNA (generally spherical) cannot enter the gel, and the relative mobility is 0 (Rm = 0), while the linear double-stranded DNA of the same size (rigid rod shape) can advance in the long axis direction. (Rm>0), it can be seen that the relative mobility of these three configurations mainly depends on the gel concentration, but at the same time, it is also affected by the current intensity, the buffer ionic strength and the like.
3, electrophoresis method
(1) Gel type
Agarose gel electrophoresis for separating nucleic acids can be classified into vertical type and horizontal type (flat type). In horizontal electrophoresis, the gel plate is completely immersed in the electrode buffer for 1-2 mm, so it is also called a submersible type. At present, the latter is used more, because it is convenient to make glue and sample, the electrophoresis tank is simple, easy to manufacture, and it can be prepared according to the need to prepare gel plates of different specifications, which saves gel and is therefore more popular.
(2) Buffer system
In the absence of ions, the current is too small and the DNA migration is slow; on the contrary, the high ionic strength buffer generates a large amount of heat due to the large current, and in severe cases, the gel melts and the DNA denatures.
Commonly used electrophoresis buffers include EDTA (pH 8.0) and Tris-acetic acid (TEA), Tris-boric acid (TBE) or Tris-phosphoric acid (TPE), and the concentration is about 50 mmol/L (pH 7.5-7.8). The electrophoresis buffer is generally formulated as a concentrated stock solution, which is diluted to the desired multiple when used.
The TAE has a low buffering capacity, and the latter two have a sufficiently high buffering capacity and are therefore more commonly used. Long-term storage of TBE concentrated solution will precipitate. To avoid this disadvantage, store 5× solution at room temperature and dilute 10 times 0.5× working solution to provide sufficient buffering capacity.
(3) Preparation of gel
Using a diluted electrode buffer as a solvent, prepare a certain concentration of sol in a boiling water bath or a microwave oven, pour into a horizontal plastic frame or a vertical film, insert a comb, and naturally cool.
(4) Sample preparation and sample loading
The DNA sample is dissolved in an appropriate amount of Tris-EDTA buffer containing 0.25% bromophenol blue or other indicator dye containing 10%-15% sucrose or 5%-10% glycerol to increase its specific gravity and concentrate the sample. To avoid sucrose or glycerol that may result in U-shaped bands for electrophoresis, 2.5% Ficoll (polysucrose) can be used instead of sucrose or glycerol.
(5) Electrophoresis
The experimental results of agarose gel separation of macromolecular DNA showed that the separation effect was better at low concentration and low voltage. Under low voltage conditions, the electrophoretic mobility of linear DNA molecules is proportional to the voltage used. However, as the electric field strength increases, the increase in mobility of larger DNA fragments is relatively small. Therefore, as the voltage increases, the electrophoresis resolution decreases. In order to obtain the maximum resolution of the DNA fragment separated by electrophoresis, the electric field strength should not be higher than 5 V/cm.
The temperature of the electrophoresis system has no significant effect on the electrophoretic behavior of DNA in an agarose gel. Electrophoresis is usually carried out at room temperature, and only when the gel concentration is less than 0.5%, in order to increase the gel hardness, electrophoresis can be carried out at 4 °C.
(6) Dyeing and photographing
Commonly used fluorescent dye ethidium bromide (EB) staining, observation of DNA bands under ultraviolet light, taking pictures with an ultraviolet analyzer, or outputting photos with a gel imaging system, and performing related data analysis.
SDS-PAGE adhesive preparation
I. Experimental principle:
SDS-PAGE is an economical, rapid, and reproducible method for quantifying, comparing, and characterizing proteins. This method is based on the difference in molecular weight of the mixed protein.
SDS is a detergent that binds to the hydrophobic portion of a protein, destroys its folded structure, and is widely present in a broadly uniform solution. The length of the SDS protein complex is proportional to its molecular weight. After adding a strong reducing agent and detergent to the sample medium and gel, the charge factor can be ignored. The mobility of the protein subunit depends on the molecular weight of the subunit.
2. Reagents and equipment:
Reagents: 1. 5x sample buffer (10 ml): 0.6 ml 1 mol/L Tris-HCl (pH 6.8), 5 ml 50% glycerol, 2 ml 10% SDS, 0.5 ml mercaptoethanol, 1 ml 1% bromophenol blue, 0.9 ml of distilled water. It can be stored at 4 ° C for several weeks or at -20 ° C for several months.
2. Gel stock solution: In the fume hood, weigh 30g of acrylamide and 0.8g of methyl bisacrylamide. After adding distilled water, dissolve it to 100ml. Filtered and placed in a brown bottle, stored at 4 ° C, generally can be placed for 1 month.
3. pH 8.9 separation gel buffer: Tris 36.3g, add 1mol / L HCl 48ml, add 80ml of distilled water to dissolve, adjust pH 8.9, dilute to 100ml, 4 ° C preservation.
4. pH 6.7 concentrated gel buffer: Tris 5.98g, add 1mol / L HCl 48ml, add 80ml of distilled water to dissolve, adjust pH 6.7, dilute to 100ml, 4 ° C preservation.
5. TEMED (tetraethylethylenediamine) stock solution
6.10% ammonium persulfate (freshly prepared with red distilled water)
7. pH8.3 Tris-glycine electrode buffer: Weigh 1.6s of Tris, 28.8g of glycine, add about 900ml of distilled water, adjust pH to 8.3, and dilute to 1000ml with distilled water. Store at 4 ° C and dilute 10 times before use.
8. Coomassie Brilliant Blue G250 staining solution: Weigh 100mg Coomassie Brilliant Blue G250, dissolve in 200ml of distilled water, slowly add 7.5ml of 70% perchloric acid, and finally make up the water to 250ml, stir for 1 hour, filter with small hole filter paper.
Equipment: electrophoresis, electrophoresis tank, water bath, shaker.
3. Experimental operation;
(1) Sample preparation
Protein samples were mixed with 5X sample buffer (20 ul + 5 ul) in an Eppendorf tube. Heat at 100 ° C for 5-10 min, take the supernatant and sample.
(2) Preparation of separation gel and concentrated gel 1 Wash the glass plate, sample comb and Spacer with detergent, rinse with ddH2O several times, then wipe with ethanol and dry;
2 Add Spacer between the two washed glass plates and install the glass plate according to the instructions of Bio-RadMini II/III.
3 Prepare 8.0 ml of 10% separation gel in the following volume and mix;
ddH2O 3.0 ml
1.0 mol/LTris-HCl pH=8.8 2.1 ml
30% Acr-Bis 2.8 ml
10% SDS 80 ul
10% AP 56 ul
TEMED 6 ul
4 Fill the glass with separate separation glue, immediately cover a layer of re-distilled water, and the glue can be polymerized after about 20 minutes;
5 Prepare 3.0 ml of 6% concentrated gel in the following volume and mix; ddH2O 1.0mol/LTris-HClpH=6.8 30% Acr-Bis
2.0 ml 400 ul 600 ul
10% SDS 10% AP TEMED
36ul 24ul 4ul
6 Pour the upper layer of distilled water, blot the filter paper, fill the concentrated gel, and insert the sample comb;
7 Install the electrophoresis system, add electrode buffer, and load 20 μl;
8 When the voltage is 200V, when bromophenol blue just runs out of the separation gel, the electrophoresis is stopped, which takes about 45 minutes to 1 hr.
9 Remove the rubber sheet, put the stripping gel into the staining solution, and dye it at room temperature for 1~2 hr; add the decolorizing solution and put it on the decolorizing shaker at 80 rpm, and replace the decolorizing solution every 20 min (10 ml glacial acetic acid; 45 ml ethanol; 45 Ml distilled water) to completely remove.
Storage Basket,Portable Plastic Bucket,Plastic Toy Packing Box,Plastic Toy Snacks Drum
Taizhou Baiying Commdity Co., Ltd , https://www.baiyingplastics.com