DNA electrophoresis (agarose gel)
DNA electrophoresis (agarose gel)
DNA electrophoresis can be used to (1) separate DNA fragments of different sizes, (2) identify target DNA fragments, and (3) purify and recover DNA fragments.
Operation method
DNA agarose gel electrophoresis
Principle
The charge effect and the molecular sieve effect that DNA molecules have when swimming in agarose gels are utilized. The charge effect refers to the fact that DNA molecules are negatively charged in a pH solution above the isoelectric point and move toward the positive pole in an electric field, and that the same amount of double-stranded DNA has almost equal net charge and can move at the same rate. Typically, the rate of DNA swimming decreases with increasing length of the DNA fragments and is proportional to the strength of the electric field. The molecular sieve effect refers to the fact that agarose, as a support medium, has a network structure that allows large molecules to receive greater resistance as they pass through, separating DNA fragments of different sizes accordingly. Generally, DNA agarose gel electrophoresis can separate DNA fragments of 50bp to millions of bp in length, and agarose gels of different concentrations and configurations are prepared according to actual needs.
Materials and Instruments
DNA Sample Move I. Range of different concentrations of standard agarose to separate DNA fragments 0.5 700bp~25kb 0.8 500bp~15kb 1.0 250bp~12kb 1.2 150bp~6kb 1.5 80bp~4kb Electrophoresis buffer preparation Take Tris-acetate and EDTA buffer (pH 8.0, TAE) as an example TAE 1X 50X 40 mmol/L Tris-acetate 242g Tris 1 mmol/L EDTA 57.1 ml glacial acetic acid 100 ml 0.5 mol/L EDTA (pH 8.0) III. Agarose gel preparation IV. DNA Electrophoresis Caveat 1. agarose: different manufacturers, different batch numbers of agarose, its impurity content is different, affecting the migration of DNA and the intensity of the fluorescent background, should be used selectively. 2. Gel preparation: the buffer added to the gel should be consistent with that in the electrophoresis tank, and the melted gel should be poured into the plate in time to avoid solidification and clumping before pouring. The gel should be poured into the plate to avoid air bubbles, so as not to affect the electrophoresis results. 3. electrophoresis buffer: in order to maintain the ionic strength and pH required for electrophoresis, the electrophoresis buffer should be updated frequently. 4. Sample addition amount: In general, 0.5cm wide comb can add 0.5 micrograms of DNA amount, the amount of sample addition is based on the size of the spiked well and the number and size of DNA fragments. When the spiking hole is large, the sample volume should be increased accordingly, otherwise it will result in shallow bands or even unrecognizable; on the contrary, the sample volume should be reduced appropriately, because too much sample volume will result in spiking hole overloading, which will lead to trailing or dispersion, for larger DNA fragments, this phenomenon is more obvious. 5. Salt concentration in DNA samples affects DNA mobility, and the same buffer conditions should be used for parallel control samples to eliminate this effect. 6. DNA mobility depends on the concentration of the agarose gel and the shape and size of the migrating molecules. It is possible to resolve a wide range of DNA molecules using gels of different concentrations, and the preparation of agarose gels can be based on the range of DNA molecules to determine the concentration of the gel. Detection of small fragments of DNA should be done by polyacrylamide gel electrophoresis to improve resolution. Common Problems Common Problems Cause Analysis 1. Run out of the DNA band blurred? (1) DNA degradation: avoid nuclease contamination. (2) Electrophoresis buffer is not fresh: after the electrophoresis buffer is used for many times, the ionic strength decreases, the pH value rises, and the buffering capacity weakens, thus affecting the electrophoresis effect. It is recommended to replace the electrophoresis buffer frequently. (3) Inappropriate electrophoresis conditions: the electrophoresis voltage should not exceed 20V/cm and the temperature should be <30℃; for giant DNA strand electrophoresis, the temperature should be <15℃; check whether the electrophoresis buffer used has sufficient buffering capacity. (4) Excessive amount of DNA samples: Reduce the amount of DNA samples in the gel. (5) DNA sample contains too much salt: remove excessive salt by ethanol precipitation before electrophoresis. (6) Protein contamination: remove protein by phenol extraction before electrophoresis. (7) DNA denaturation: do not heat the sample before electrophoresis and dilute the DNA with 20 mM NaCl buffer. 2. There is irregular DNA band migration? (1) For λ/Hind III fragment cos site recombination: heat DNA at 65℃ for 5 minutes before electrophoresis, then cool on ice for 5 minutes. (2) Inappropriate electrophoresis conditions: electrophoresis voltage should not exceed 20V/cm, temperature <30℃; change the electrophoresis buffer frequently. (3) DNA denaturation: do not heat before electrophoresis, dilute DNA with 20mM NaCl buffer. 3. run out of DNA bands weak or no bands? (1) The amount of DNA sample is not enough: increase the amount of DNA sample. (2) DNA degradation: avoid nuclease contamination of DNA. (3) DNA out of the gel: connect the electrodes correctly to avoid inserting the opposite, shorten the electrophoresis time, reduce the voltage, and enhance the gel concentration. (4) Inappropriate light source: For DNA stained with EB, use a short wavelength (254nm) ultraviolet light source. 4. run out of DNA bands missing incomplete? (1) If it is a small DNA band, it may run out of the gel, you can shorten the electrophoresis time or reduce the voltage or enhance the gel concentration. (2) DNA bands of similar molecular size are not easy to distinguish, electrophoresis time should be extended and the correct gel concentration should be approved. (3) DNA denaturation: do not heat before electrophoresis and dilute the DNA with 20 mM NaCl buffer. For more product details, please visit Aladdin Scientific website.
Agarose Electrophoresis Buffer Ethidium Bromide Sample Buffer
Electrophoresis Instrument Electrophoresis Tank Transmission UV Lamp Tape Paper UV Imager
Agarose/% Range of resolving DNA size
Buffer Working solution Storage solution/L
1. Place a clean and dry plastic tray into the gel dispensing plate and place it on a horizontal surface.
2. Prepare a sufficient amount of electrophoresis buffer (1XTAE) to fill the electrophoresis tank and prepare the gel. 3.
3. According to the size of the DNA fragments to be separated, prepare the appropriate concentration of agarose solution with the electrophoresis buffer: accurately weigh the dry agarose powder and add it to the triangular flask or glass bottle containing a fixed amount of electrophoresis buffer.
4. Heat in a boiling water bath or microwave oven until the agarose melts. 5.
5. Transfer the triangular flask or glass vial to a 55°C water bath using insulated gloves or clamps. Allow the melted gel to cool slightly and then add ethidium bromide to a final concentration of 0.5 μg/ml. swirl gently to mix the gel solution well.
6. While the agarose solution is cooling, use a suitable comb to form the spiking wells. The teeth of the comb should be positioned 0.5 to 1.0 mm above the bottom surface of the tray so that the agarose will form the desired spiking holes when poured into the tray.
7. Pour the warm agarose solution into the mold and leave it at room temperature for 30~45min. When the gel solution is completely solidified, pull out the comb carefully.
1. Place the gel in the electrophoresis tank, add electrophoresis buffer to the electrophoresis tank, just missing the gel about 1mm.
2. Mix the DNA sample and a certain proportion of the loading buffer. 3.
3. Use a micropipette and disposable tip to slowly add the sample mixture to the sample wells that submerge the gel. The molecular mass standard should be added to the left and right of the sample wells.
4. Close the lid of the electrophoresis tank and connect the electrode plug. the DNA should swim towards the anode (red plug) side. Give 1~5V/cm where the distance is measured between the anode and cathode.
5. when the DNA sample or dye has migrated a sufficient distance in the gel, turn off the power, unplug the electrodes and open the lid of the electrophoresis tank. observe the gel with a UV lamp and take photographs.
5. Experimental Methods Sources Sources Guide to Molecular Cloning Experiments.