CBS denaturing gradient gel electrophoresis experimental operation
ã€Purpose】 [Experimental principle] [Instruments, Materials and Reagents] [Experimental steps] ã€Precautions】 Dehydrated Scallion,Dehydrated Spring Onion Rings,Dehydrated Scallion Powder,Dried Green Scallion Xinghua Lvwei Foods Co.,Ltd , https://www.lvweifoods.com
Master the novel mutations in denaturing gradient gel electrophoresis and the technical methods for determining genotypes of highly polymorphic genes.
The analysis of DNA sequence mutations in modern genetics plays an important role. Since it is very difficult to detect a subtle mutation in a large DNA sequence, several methods have been established to solve this problem. Denaturing gradient gel electrophoresis (DGGE) separates double-stranded DNA fragments of the same length and different nucleotide sequences . This method takes advantage of the fact that the electrophoretic mobility of a DNA molecule decreases from a double helix to a localized type. The gradients required for this change in different DNA fragments are different. In the DGGE gel, the content of the denaturant ( formaldehyde and urea ) in the direction of the electric field is increased. When the DNA fragment is passed through the gel of the denaturant, the electrophoretic mobility of the different molecules decreases in different regions. This allows the nucleotide sequences to be separated by different DNA fragments. This method can be used as an initial step in sequencing to isolate alleles in heterozygous individuals. Many studies have shown that delivery of denatured gel electrophoresis separation ability is very strong, it can be separated only difference 1bp DNA fragments.
1.50×TAE buffer (2 mol/LTris acetate, 0.05 mol/L EDTA pH 8.0) 1 L volume: 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 mol/L EDTA pH 8.0 , add water to lL .
2. Acrylamide stock solution: 40% acrylamide (38: 2 acrylamide : bis acrylamide ) .
3. Ammonium persulfate stock solution (10%) : 10mL preparation: 1g ammonium persulfate plus water to 10mL . TEMED (N , N , N ', N ', - tetramethylethylenediamine ) .
4. Denaturing agent stock solution: (0%) 6% acrylamide TAE solution. 250 mL solution: 37.5 mL acrylamide stock solution, 5 mL 50×TAE buffer, add water to 250 mL , filter and vent.
5. Denaturing agent stock solution (100%) : 6% acrylamide, 7 mol/L urea, 40% formaldehyde TAE solution. 250 mL preparation: 37.5 mL acrylamide stock solution, 5 mL 50×TAE buffer, 105 g urea, 100 mL formaldehyde, add water to 750 mL , filter and vent.
6. Dyes: 40% (w/v) sucrose, 0.25% bromophenol blue, 0.25% xylene cyanide and 30% glycerol.
7. Plastic frame, comb and gasket, two plastic brackets for fixing the glass in the plastic frame.
8. A glass plate with a handle and a handleless ( dimensions: 17.78 cm wide x 20.32 cm long x 0.635 cm thick ) , the stalked glass plate has a 13.97 cm wide, 2.54 cm thick projection .
9. Plastic groove without metal structure ( size: 43.18cm long × 22.86cm wide × 22.86cm deep; one or two pieces of glue can be allowed to electrophoresis at the same time ) .
10. Power supply, anode - platinum; cathode - graphite rod ( diameter 0.635cm) .
11. Peristaltic pump and connecting pipe, stirrer and heater, gradient generator: 15 ~ 25mL per side volume .
1. Preparation of Parallel Glue: The concentration of the denaturant from top to bottom in the parallel gel is increased. This glue is suitable for analyzing large numbers of samples.
1) A rubber sheet was prepared in the same manner as a standard polyacrylamide gel.
2) Take two equal volumes of solution from acrylamide and two denaturing agent stocks to meet the desired denaturant concentration range. The total volume of the solution is generally just covered with a rubber sheet. Acrylamide concentration depending on the size of the DNA fragments, for DNA fragment size ≥300bp acrylamide concentration was 6%, the length <300bp DNA fragment, the acrylamide concentration was 12%.
3) Add the high concentration denaturing agent solution to the right tank of the gradient generator ( the solution will first enter the gap between the rubber sheets through the gradient synthesizer ) , open the switch connecting the two ends of the gradient generator, and let some solution flow into the left tank to close the switch. , use a straw to move them back to the right slot (the purpose is to ensure no bubble barrier between the two slots ) . The low concentration denaturing agent solution is then added to the left tank of the gradient generator.
4) stirring the two tank solution is added thereto 4.5μL / mLl0% ammonium persulfate and 1μL / mL of TEMED (N, N, N ' , N', - tetramethylethylenediamine).
5) Gently open the switch between the two slots and the switch at the outlet of the gradient generator. The solution in the two tanks should be stirred during the filling ( especially the high concentration solution tank should be stirred ) , and the solution is placed by gravity. A thin plastic tube at the top between the two glass plates flows through the gradient generator. The flow rate should be kept constant to ensure that all solutions should be injected between the two glass plates before the solution is agglomerated. (The rate of condensation can be adjusted by adjusting the concentration of ammonium persulfate and TEMED )
6) After the rubber sheet is full, insert the comb at the top and flatten it for at least 30 minutes . (The glue can be filled beforehand and stored at 4 ° C for a few days ) .
2. Preparation of vertical glue: The vertical glue contains a concentration of acrylamide that is fixed, with a linearly increasing gradient of denaturant perpendicular to the direction of electrophoresis. This glue is useful for determining the optimal gradient range for isolating DNA fragments containing nucleotide changes . The preparation of vertical glues is generally similar and the preparation of parallel glues, but also differs:
1) A rubber sheet is prepared like a standard polyacrylamide gel, but no comb is inserted at the top.
2) Prepare a lubricant-coated spacer that is slightly shorter than the comb and insert it in the position of the comb. The position is slightly protruding so that a gap is left on the left side. Clamp this side with a clip. Rotate the rubber plate 90° so that the face connected to the notch is turned to the top. The glue will pass between the two glass sheets through the gap. The method is as described above.
3) After the gel is completely solidified, the rubber plate is erected vertically, and the spacer at the top of the glue is gently removed. Large, flat holes appear on the top of the gel, allowing electrophoresis.
3. DNA electrophoresis: ( parallel or vertical DGGE steps are the same )
1) After removing the top spacer or comb, transfer the relevant equipment and glue to the electrophoresis tank containing the buffer heated to 60 ° C so that the buffer height has not just passed through the sample well on the gel.
2) Connect the peristaltic pump to allow the buffer to flow from the tank to the top tank ( cathode ) . The buffer then flows back through the pores at the edge of the frame back into the tank ( top tank buffer circulation is necessary to avoid a significant increase in pH and changes in ionic strength during electrophoresis ) .
3) For vertical gel electrophoresis, add 100 μL of PCR product ( including 20 μL of loading dye ) to the well of the top of the gel ; for parallel gel electrophoresis, add 15-20 μL of sample to each well. .
4) Place the graphite electrode in the top groove according to the gel width, and electrophoresis for 3 to 15 hours at a voltage of 150 to 350 V according to the size of the fragment . On the parallel gel, 250V , electrophoresis for 5h can move the 300bp fragment to 1/3 of the gel .
5) Staining was observed in 0.5 μg/mL ethidium bromide and observed.
1. Avoid creating bubbles during mixing and filling.
2. Sometimes there is a shrinkage on the left side of the polyacrylamide gel so that an air passage is created between the top and bottom of the gel, which can be filled with a 2% molten agarose gel.
3. All solutions should be stored in a 4 ° C brown bottle, usually valid for several months to one year.
4. The gel temperature must be kept constant during electrophoresis. To meet this requirement, the rubber sheet can be immersed in a well-stirred temperature-controlled buffer tank. For DNA that is more susceptible to denaturation in the absence of a denaturant , the temperature in the tank is chosen to slightly exceed the melting point at 60 ° C , and most of the work is carried out at 60 ° C ( but a slightly higher or lower temperature can be used ) . The temperature is kept constant. The electrophoresis buffer can be heated to 60 ° C and the gel can be immersed therein for electrophoresis.
Related links: http://