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Methodology for Nucleic Acids

 
     
 
DNA Electrophoresis in Agarose Gel
 
Many different techniques are available for the fractionation and characterization of nucleic acids including the recombinant DNA. Electrophoresis of DNA or RNA in gels is a rapid and relatively inexpensive method and can be easily set up in any laboratory. DNA can be checked for size, intactness, homogeneity and purity by this technique. Importantly, this technique is central to gene manipulation experiments.
 
Separation of DNA can be achieved in polyacrylamide or most preferably in agarose gels. Agarose vertical gels give reproducible results and can be completed more rapidly than many horizontal systems. Horizontal agarose gel electrophoresis has the advantage that much lower concentration of agarose can be utilized than in a vertical gels system allowing the separation of large DNA fragments, and also handling of the gel is easier.
 
An effective compromise between the standard vertical and horizontal slab gel is the 'submarine minigel’. These small (10 X 6 X 0.5cm) agarose gel slabs are run horizontally under approximately 2-5 mm buffer and have the advantages of being easier to prepare and faster-running than the conventional gels.
 
 



Principle

Agarose forms a gel by hydrogen bonding and the gel pore size depends on the agarose concentration. The DNA molecules are separated by electrophoresis on the basis of their size, shape and the magnitude of net charge on the molecules. The dye ethidium bromide intercalates between the bases of RNA and DNA and fluorescences orange when irradiated with UV light. Low concentration agarose gels with large pore permit fractionation of high MW molecules and vice versa.
 
 



Materials

Tris-Borate Buffer    (10X; pH8.2)
   0.9M Tris-HCl                    113.0g
   0.025M EDTA Na2            9.3g
   0.9M boric acid                 55g
   Double distilled water      1L

Agarose. 1.0 (wlv) in single-strength Tris-borate (gel-running) buffer.
   Autoclave to dissolve the agarose; then maintain at 50°C in a stoppered flask until used.

Gel-loading Solution:
   Sucrose 30%, Bromophenol blue 0.25% and Xylene cyanole FF 0.25% (all wlv) in single-strength Tris-borate buffer.
DNA Preparations (Plant DNA, plasmid DNA, recombinant DNA etc.)
Standard DNA for Size Determination
   (l DNA cut with restriction endonucleases Eco RI and Hind III)
Gel Casting Plate
   Gel tank, power pack etc.
UV transilluminator with photography system, safety glasses
 
 
Procedure
1.
Form a wall around clean dry, gel-casting glass plate (10 X 6 x 0.5cm) using zinc oxide tape. This should give a leakproof wall about 1cm high all around the plate. Alternatively, the plate is placed in a suitable gel casting tray purchased from a commercial supplier. Place the set-up perfectly horizontal over a leveled plate.
2.
Pour 30mL of 1% agarose solution maintained at 50°C onto the casting plate. Immediately place a suitable well-forming comb about 1cm from one end of the plate.  The teeth of comb should not touch the glass plate. Allow the gel to set for 1h.
3.
Remove the comb from the gel, carefully. Transfer the gel along with the glass plate to the electrophoresis tank such that the wells are near the cathode. Pour single-strength Tris-borate buffer into the tank until the gel (including zinc oxide wall) is submerged.
4.
Connect the electrodes to the power supply with the cathode (-ve) at the well end of the gel.
5.
Load DNA samples (5-20//L) and standard taken in gel-loading solution using a microsyringe.
6.
Turn on the power supply and run at 100V (10-15mA). Monitor the progress of fast-running (bromophenol blue) tracking dye during electrophoresis. Terminate the run when the tracking dye is about to leave the gel.
7.
After disconnecting the power supply, transfer the gel to a staining tray containing 250/ig ethidium bromide (50//L of 5mg/mL solution) in 250mL of used Tris-borate buffer. Stain the gel for about 30 min.
8.
Transfer the gel onto a thick plastic sheet, place on a UV transilluminator and view the gel under ultraviolet light (300nm). Nucleic acids on the gel will appear orange owing to the fluorescence of bound ethidium bromide. Photograph as soon as the gel has been checked for the presence of bands. Use the photograph for further interpretation of band patterns.
9.
Measure (from photograph) the distance moved by each band from the front of the loading well. Plot the distances against log molecular weight of standards (A DNA fragments) to give a calibration curve. Deduce the size of DNA and/or restricted fragments of samples using the curve.
 
 
Notes
1.
Agarose used for electrophoresis should be of very high quality - low sulphate content and electroendosmosis (e.g., Sigma Type I grade).
2.
Agarose gels of different thickness, concentration (0.2-2%) and dimensions are used depending upon the experimental requirement.
3.
Pour the gel carefully without entrapping any air bubbles. Keep the agarose solution always at 50-55°C until poured on the casting plate to avoid setting. Handle the gel carefully as it is delicate.
4.
Minigels are run at high current for quick results. Otherwise, the gels of large size are usually run at a constant low voltage (25-40) overnight for high resolution of bands.
5.
Wear gloves while using ethidium bromide as it is mutagenic.
6.
Ethidium bromide may be added to the gel running buffer itself. The separation of bands then can be followed even during electrophoresis using a hand-held UV lamp.
7.
Wear safety glasses for viewing the gel under UV light.   In order to prolong the life of the transilluminator and to avoid excessive exposure to UV radiation, photograph the gel at the shortest period and use the photograph for further interpretation. Position a mini-scale and photograph to measure the relative mobilities of bands later.
8.
The electrophoretic mobility of bands is very much affected by the salt concentration in the sample. It is important that the salt concentration in samples loaded in various wells is (nearly) uniform.
9.
The sizes of restriction fragments from lambda DNA (49kb) are given below:
                 Sizes of Restriction Fragments from X DNA (49kb)
 
Size, kilobase pairs (kb)
 
EcoRI
Hind III
Eco RI plus Hind III
21.80
23.70
21.80
7.52
9.46
5.24
5.93
6.75
5.05
5.54
4.26
4.21
4.80
2.26
3.41
3.41
1.98
1.98
 
 
1.90
 
 
1.71
 
 
1.32
 
 
0.93
 
 
0.84
 
 
0.58
 
 
References
1.  Maniatis, T, Fritsch, E F and Sambrook, J (1982) Molecular cloning: A Laboratory Manual Cold Spring Harbour Laboratory New York.
2.  Rodriguez, R L and Tait, R C (1983) Recombinant DNA Techniques: An Introduction Benjamin Cummings Pub Co Inc London.
3.  Manual on 'Techniques in Molecular Biology' (Nucleic Acids) (1986) Workshop held at The Department of Biochemistry Tamil Nadu Agricultural University Coimbatore p 14.
 
     
 
 
     




     
 
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