Agarose Gel Electrophoresis

From 2007.igem.org

(Difference between revisions)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
It is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).  
It is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).  
-
Applications
+
Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.
-
    * Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of     cloned DNA.
+
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.
-
    * Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
+
-
    * Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.
+
-
The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered.
+
Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are:
-
The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.
+
Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.
-
Preparation
+
-
There are several methods for preparing agarose gels. A common example is shown here. Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a constant amount while length and breadth are varied as needed). The vast majority of agarose gels used in modern biochemistry and molecular biology are prepared and run horizontally.[citation needed]
+
The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.
 +
-
  1. Make a 2% agarose solution in 100ml TAE. A solution of up to 4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.8% is preferable. Use 15-100 mL, depending on the size of the gel.
+
Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).
-
  2. Bring the solution to the boil to dissolve the agarose, preferably in a microwave oven.
+
-
  3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or swirl the solution while cooling.
+
-
Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety see ethidium bromide
+
Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.
-
  1. Add 5 µl ethidium bromide stock per 100 ml gel solution. Be very careful when handling the concentrated stock. Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
+
Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.
-
  2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
+
-
  3. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
+
-
  4. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots.
+
-
  5. Put the gel, together with the rack, into a tank with 0.5x TBE. Ethidium bromide at the same concentration can be added to the buffer. Make sure the gel is completely covered with TBE, and that the slots are at the end electrode that will have the negative current.[citation needed]
+
-
[edit] Procedure
+
[[Image:Gel.jpg|250px]]                                                                            [[Image:Gel2.jpg|250px]]
-
 
+
-
After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. It is
+
-
now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light. [citation needed]
+
-
 
+
-
Steps: [citation needed]
+
-
 
+
-
  1. The agarose gel with three slots/wells (S).
+
-
  2. Injection of DNA ladder (molecular weight markers) into the first slot.
+
-
  3. DNA ladder injected. Injection of samples into the second and third slot.
+
-
  4. A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone.
+
-
  5. Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.
+
-
  6. Add the color marker dye to the DNA ladder.
+

Latest revision as of 11:06, 21 October 2007

It is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.


Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.


Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

Gel.jpg Gel2.jpg

Retrieved from "http://2007.igem.org/wiki/index.php/Agarose_Gel_Electrophoresis"