DNAElectrophoresis

发布时间：2019-08-08 23:36 原文链接： DNAElectrophoresis

What is Electrophoresis?

Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.

What is a Gel?

Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like "Jello" only more firm.
The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose).

DNA Electrophoresis
Agarose gel electrophoresis can be used to separate DNA molecules according to size. In fact, we load a dye with our DNA samples so that we can monitor the progress of the electrophoresis. The dye solution also contains glycerol to provide a dense mixture that will stay in the bottom of the well.

Materials

DNA samples
DNA size standard
Loading dye solution
Agarose
TBE buffer
Sybr Green I solution diluted 10,000-fold
Dark Reader light box
Plastic wrap

Procedure

::Pour an agarose gel and cover it with TBE buffer.::Pipette 5 ul drops of the loading dye solution onto a piece of plastic wrap (one for each DNA sample)::Pipette 5 ul of each DNA sample into one of the drops of loading dye. Mix by pipetting up and down and carefully load into a well of the agarose gel.::Pipette 10 ul of a solution containing a DNA size standard into an empty well of the gel.::Place the lid on the gel box and turn on the power supply to 100 volts.::After the tracking dye has migrated half to two thirds of the way through the gel, turn off the power, remove the gel from the gel box and place in a tray containing enough Sybr Green I solution to cover the gel. Swirl occasionally. After 15 minutes, remove the gel from the staining solution and place on the light box. Observe the gel through the orange filter in a dimly lit room.::Save the Sybr Green I solution. It can be reused several times.

10 Fun Facts of DNA Electrophoresis

::When preparing agarose for electrophoresis, it is best to sprinkle the agarose into room-temperature buffer, swirl, and let sit at least 1 min before microwaving. This allows the agarose to hydrate first, which minimizes foaming during heating.::Electrophoresis buffer can affect the resolution of DNA. TAE (Tris-Acetate-EDTA) buffer provides better resolution of fragments >4 kb, while TBE (Tris-Borate-EDTA) buffer provides better resolution of 0.1- to 3-kb fragments. In addition, use TBE buffer when electrophoresing >150 V and use TAE buffer with supercoiled DNA for best results.::Migration of DNA is retarded and band distortion can occur when too much buffer covers the gel. The slower migration results from a reduced voltage gradient across the gel..::Loading DNA in the smallest volume possible will result in sharper bands.::You can preserve DNA in agarose gels for long-term storage using 70% ethanol. [See Jacobs, D. and Neilan, B.A. (1995) BioTechniques 19, 892.]::Electrophoresing a gel too "hot" can cause the DNA to denature in the gel. It can also cause the agarose gel to deform. Cool the gel with a small fan during the electrophoresis.::For the Supercoiled DNA Ladder electrophoresed on <1% agarose gels, add 2 1lg/ml ethidium bromide to the gel. Otherwise, smeared bands and extra bands will be seen because of different degrees of supercoiling. [See Longo, M.C. and Hartley, J.L. (1986) ]::When glycerol-containing loading buffers are used in DNA samples electrophoresed through acrylamide gels, smiling bands may be accentuated especially in TBE.::On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size. This anomalous migration is enhanced at lower temperatures and disappears at high temperatures. This anomalous migration is not observed on agarose gels. [See Stellwagen, N.C. (1983) Biochemistry22, 6186.]::The minimum amount of DNA detectable by ethidium bromide on a 3-mm-thick gel and a 5-mm-wide lane is 1 ng. Do not exceed 50 ng of DNA per band on a 3-mm-thick gel and 5-mm-wide lane.

Troubleshooting DNA Agarose Gel Electrophoresis

If you see faint or no bands on the gel:

::There was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don't exceed 50 ng/band.::The DNA was degraded. Avoid nuclease contamination.::The DNA was electrophoresed off the gel. Electrophorese the gel for less time, use a lower voltage, or use a higher percent gel.::Improper W light source was used for visualization of ethidium bromide-stained DNA. Use a shortwavelength (254 nm) W light for greater sensitivity. Note: For preparative gels, using a longer wavelength (312 nm) W light will minimize DNA degradation.

If you see smeared DNA bands:

::The DNA was degraded. Avoid nuclease contamination::Too much DNA was loaded on the gel. Decrease the amount of DNA.::Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. This is done by checking the pH in the anode and cathode chambers.::There was too much salt in the DNA. Use ethanol precipitation to remove excess salts, prior to electrophoresis.::The DNA was contaminated with protein. Use phenol extractions to remove protein prior to electrophoresis.::Small DNA bands diffused during staining. Add the ethidium bromide during electrophoresis.

If you see anomalies DNA band migration:

::Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity.::The DNA was denatured. Do not heat standards [except for l DNA/Hind III fragments (figure 1)] prior to electrophoresis. Dilute DNA standards in buffer with 20 mM NaCl.

FIGURE 1. The effect of ionic strength on the heat denaturation of l DNA/Hind III fragments. 500 ng DNA/Hind III fragments in NaCl concentrations of 0, 1, 2, 5, 10, 20, 30, and 40 mM were electrophoresed after heating at 65° C for 10 min.

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