Nucleic Acid Electrophoresis Protocols & Introduction
Introduction
Agarose gel electrophoresis for DNA
Agarose gel electrophoresis for RNA
Polyacrylamide gel electrophoresis for DNA
Reference
Introduction - What is Electrophoresis?
Electrophoresis is a method of separation and purification of macromolecules such as nucleic acids (DNA and RNA) and proteins based on the net charge, size, and conformation on a matrix. Nucleic acids have an overall negative charge due to the presence of phosphate backbone. Therefore, they move towards the anode at the migration rate that depends solely on their size. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards a pH called isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform charge, it is possible to separate them based on the size. The macromolecules are electrophoresed within a matrix or gel made up of agarose or polyacrylamide. The gels made of these polymers contain pores through which the proteins and nucleic acids can pass when voltage is applied.
Agarose Gel Electrophoresis for DNA
Agarose is a polysaccharide extracted from seaweed and is used typically at concentrations 0.5 – 2% for electrophoresis of DNA and RNA. It forms a lattice with suitable pore size that allows the movement of nucleic acids to the positive electrode.
Figure 1. Agarose gel electrophoresis of nucleic acids
Protocol: Agarose Gel Electrophoresis for DNA
Materials and Reagents Required
- Electrophoresis chamber with power supply, casting trays and combs
- Bionic™ Buffer (B6185) or an appropriate electrophoresis running buffer:
1X TAE (65497) containing:
0.04 M Tris-acetate (pH 7.6)
0.001 M EDTA
OR
1X TBE (93290) containing:
0.13 M Tris (pH 7.6)
45 mM boric acid
2.5 mM EDTA
- Loading buffer (G7654, G2526) or prepared in 1X TAE or 1X TBE with the following:
50% glycerol
0.25% bromophenol blue (B5525)
0.25% xylene cyanol FF (X4126)
- BlueView™ Nucleic Acid Stain (T9060 and T8935), SYBR® Green Nucleic Acid Stain (S9430) or ethidium bromide: 0.5 µg/mL prepared in distilled water.
- Transilluminator
Precast agarose gels
Sigma-Aldrich offers precast agarose gels in 8-, 20- and 24-well format with added ethidium bromide. Continue to “Running the gel” step if precast gels are being used.
Alternatively, cast your own agarose gels using the following procedure.
Preparation of Agarose Solution
- Measure the appropriate amount of agarose powder and add to 1X TAE buffer in a beaker or flask.
Note: The amount of agarose will depend on the percentage of the gel required. The volume of agarose solution must be prepared according to the size of the gel tray used.
- Dissolve the agarose by heating on a magnetic hot plate. Alternatively, it can be heated in a microwave, swirling every minute till the agarose is completely dissolved.
- When the agarose is cooled to 50-60 °C, add ethidium bromide (0.5 µg/mL final concentration) to the solution. Another alternative is to immerse the gel in ethidium bromide solution after electrophoresis.
IMPORTANT: Ethidium bromide is a carcinogenic agent. Always wear gloves and mask when handling ethidium bromide.
- While the agarose is cooling, prepare the gel tray for gel casting so that the agarose solution does not flow out before setting. This can be ensured by using tray dams, flexicaster or sealing with traditional laboratory tape. Place the comb in the grooves and place the tray in an even horizontal position.
- Pour the agarose solution slowly so that it is evenly distributed on the tray with no air bubbles trapped in the gel. Allow the gel to solidify at room temperature.
- Remove the combs and transfer the gel with the tray to the main tank and fill with 1X electrophoresis buffer until the gel is just covered with buffer.
Preparation of DNA Samples
Sigma-Aldrich offers GenElute™ kits for isolation of DNA from plants and fungi (E5038), mammalian cells or tissue (G1N70, G1N10 and G1N350) and blood (NA2010 and NA2020).
To protect the isolated DNA from degradation, it is recommended that the DNA be solubilized in TE buffer (T9285).
Additionally, DNAstable® kits (93000-001-1EA, 93021-001-1EA, 53091-016-2ML and 93121-017-1EA) may be used in case the DNA is being shipped or for storage and stabilization of DNA at room temperature.
- Isolate DNA from cells or tissue by the standard protocol.
- The minimum concentration of DNA required for detection on agarose gel when stained with ethidium bromide is 2 ng.
- Mix the nucleic acid samples with 10X loading buffer. Generally, 3 µL of the loading buffer is sufficient but lesser volume may be used for samples less than 10 µL.
Running the Gel
- Load the samples carefully into the wells using pipettes. A suitable marker containing nucleic acid fragments of various sizes may also be loaded (D7058, D3937, D3812).
- Place the lid on the tank and connect to the power supply. The typical voltage to run the samples in agarose gels is 90 - 150 V.
- You should track the dye front using Orange G (O3756), Bromophenol Blue (B8026), or Xylene Cyanol (x4126).
Gel Staining and Viewing
Gels incorporated with ethidium bromide:
- After electrophoresis, transfer the gel to UV transilluminator and acquire the image of the gel.
- The samples will appear as bright bands.
Gels not incorporated with ethidium bromide:
- Transfer the gel after electrophoresis into SYBR® Green Nucleic Acid Stain (diluted 1:10,000, stain in dark) or 0.5 µg/ml ethidium bromide staining solution for 15-30 min.
- If SYBR® Green Nucleic Acid Stain is being used the image of the gel may be acquired immediately after staining.
- If ethidium bromide is being used destain the gel in distilled water for 10-30 min ensuring that the gel is completely immersed in water.
- Transfer the gel to UV transilluminator and acquire the image of the gel.
- The samples will appear as bright bands.
Fluorescent Staining of Agarose Gels
Sigma-Aldrich offers Nancy-520 (01494), a fluorescent stain that can be used in the place of ethidium bromide. It is a safer, stable and more environmental-friendly alternative to ethidium bromide. Nancy-520 has an excitation wavelength of 520 nm and an emission wavelength of 560 nm.
Gels incorporated with Nancy-520:
- Nancy-520 can be incorporated into the agarose when casting the gel (10 µL to 50 mL agarose).
- After the run, acquire a fluorescence image of the gel.
Gels not incorporated with Nancy-520:
- Prepare staining solution by adding 10 µL of Nancy-520 to 50 ml of 1X TBE buffer.
- After the electrophoresis, immerse the gel in the staining solution for 1 h in the dark over a rocking table.
- Rinse the gel with 1X TBE buffer for 10-30 sec.
- Take a fluorescence image of the gel.
Figure 2.Horizontal Electrophoresis System
Agarose Gel Electrophoresis for RNA
The quality of RNA can be assessed by agarose gel electrophoresis that resolves RNA based on the size and integrity. However, RNA forms various secondary structures due to extensive intramolecular base pairing that interferes with size-based migration on the agarose gel. Therefore, the RNA molecules must be denatured using formamide and formaldehyde.
Protocol: Agarose Gel Electrophoresis for RNA
Materials and reagents required
Ensure that all the equipment used is RNAses free and all the buffers used are made in RNase-free water.
- Electrophoresis chamber with power supply, casting trays and combs
- 1X MOPS-EDTA-Sodium Acetate (MESA) buffer (M5755):
40 mM MOPS
10 mM sodium acetate
1 mM EDTA (pH 8.3)
- RNA sample buffer (R1386 and R4268)
62.5% (v/v) deionized formamide
1.14 M formaldehyde
1.25X MESA buffer
200 µg/mL bromophenol blue (B2225)
200 µg/ml xylene cyanol FF (X4126)
Sigma-Aldrich offers RNA sample buffers with (R1386) or without ethidium bromide (R4268)
- SYBR® Green Nucleic Acid Stain (S9430) or ethidium bromide: 0.5 µg/mL prepared in distilled water
- Transilluminator
Precast Agarose Gels
Sigma-Aldrich offers precast 1.25% agarose gel for RNA (P6222) in 8-well format. This precast gel does not contain ethidium bromide. Continue to “Running the gel” step if precast gels are being used.
Alternatively, cast your own agarose gels using the following procedure.
Preparation of Agarose Solution
- Measure the appropriate amount of agarose powder and add to 1X MESA buffer in a beaker or flask.
Note: The amount of agarose will depend on the percentage of the gel required. The volume of agarose solution must be prepared according to the size of the gel tray used.
- Dissolve the agarose by heating on a magnetic hot plate. Alternatively, it can be heated in a microwave, swirling every minute till the agarose is completely dissolved.
- When the agarose is cooled to 50-60 °C, add ethidium bromide (0.5 µg/mL final concentration) to the solution. Another alternative is to immerse the gel in ethidium bromide solution after electrophoresis.
IMPORTANT: Ethidium bromide is a carcinogenic agent. Always wear gloves and mask when handling ethidium bromide. - While the agarose is cooling, prepare the gel tray for gel casting so that the agarose solution does not flow out before setting. This can be ensured by using tray dams, flexicaster or sealing with traditional laboratory tape. Place the comb in the grooves and place the tray in an even horizontal position.
- Pour the agarose solution slowly so that it is evenly distributed on the tray with no air bubbles trapped in the gel. Allow the gel to solidify at room temperature.
- Remove the combs and transfer the gel with the tray to the main tank and fill with 1X electrophoresis buffer until the gel is just covered with buffer.
Preparation of RNA Samples
- Isolate RNA from cells or tissue samples using the TRI Reagent® (T9424) or GenElute™ kit for mammalian cells or tissues (RTN70, RTN10 and RTN350).
- To determine the quality and concentration of RNA, read the absorbance of the samples at 260 nm and 280 nm. The ratio of absorbance, A260/A280 of 1.8-2.1 indicates good quality RNA. For effective detection by agarose electrophoresis, at least 5 µg of RNA is required.
- Mix one volume of RNA sample with 2-5 volumes of sample buffer.
- Heat the sample to 65 °C for 10 min and chill immediately on ice to prevent renaturation of RNA molecules.
Running the Gel
- Load the samples carefully into the wells using pipettes. A suitable marker containing RNA fragments of various sizes (R7020, R7644) may also be loaded, if required.
- Place the lid on the tank and connect to the power supply. The typical voltage to run the samples in agarose gels is 90 - 150 V.
- You should track the dye front using Pyronin Y (P9172), Bromophenol Blue (B8026), or Xylene Cyanol (x4126).
Gel Staining and Viewing
Gels incorporated with ethidium bromide:
- After electrophoresis, transfer the gel to UV transilluminator and acquire the image of the gel.
- The samples will appear as bright bands.
Gels not incorporated with ethidium bromide:
- Wash the gel in RNase free distilled water gently for 10 min to remove formaldehyde.
- Transfer the gel after electrophoresis into SYBR® Green Nucleic Acid Stain (1:10,000) or 0.5 µg/ml ethidium bromide staining solution for 15-30 min.
- If SYBR® Green Nucleic Acid Stain is being used the image of the gel may be acquired immediately after staining.
- If ethidium bromide is being used destain the gel in distilled water for 10-30 min ensuring that the gel is completely immersed in water.
- Transfer the gel to UV transilluminator and acquire the image of the gel.
- The samples will appear as bright bands.
Polyacrylamide Gel Electrophoresis for DNA
Polyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix. Acrylamide gels can separate DNA fragments that differ by even 0.2% in length. While proteins must be denatured by SDS before separation on polyacrylamide, DNA molecules being negatively charged need not be denatured. In comparison with agarose gels, polyacrylamide gels can accommodate larger amounts of samples and provide high resolution of DNA fragments.
Although precast PAGE gels for DNA are popularly used the cost involved is high if PAGE is a regular procedure in the laboratory. The skilled technician can prepare PAGE gels with little effort at a fraction of cost of precast gels. PAGE gels prepared in-house provide better resolution and aid in achieving consistent results.
Protocol: Polyacrylamide Gel Electrophoresis for DNA
Materials and Reagents Required
Vertical electrophoresis chamber with power supply, glass plates, spacers and combs
- 30% polyacrylamide solution (filter through 0.45 mM filter and store in the dark at 4 °C):
29 g Acrylamide
1 g Bis-acrylamide
100 mL double-distilled water
- 10% Ammonium persulphate solution
- TEMED
- Electrophoresis buffer:
1X TBE prepared in distilled water:
89 mM Tris (pH 7.6)
89 mM boric acid
2 mM EDTA
- 5X gel loading buffer:
80% glycerol 75%
Bromophenol blue (B5525) 0.25%
Xylene cyanol (X4126) 0.25%
1M Tris (pH 7.4) 10 mM
5 M NaCl 10 mM
0.5 M EDTA 10 mM
10% SDS 0.1%
- Ethidium bromide 0.5 µg/ml
- Transilluminator
IMPORTANT: Acrylamide and bis-acrylamide are neurotoxic in nature. All the steps should be performed wearing powder-free gloves.
Procedure
- Clean the glass plates and spacers of the gel casting unit with deionized water and ethanol.
- Assemble the plates with the spacers on a stable surface.
- Prepare gel solution using the following volumes (for 12 mL) depending on the percentage of gel required.
*TEMED must be the last ingredient added.
- Pour the gel solution in the plates assembled with spacers. Insert the comb immediately ensuring no air bubbles are trapped in the gel or near the wells. Allow the gel to set for about 30-60 min at room temperature
The polymerized gel may be wrapped in Saran wrap and stored at 4 °C for future use.
- When ready to perform electrotrophoresis, mount the plates with gel in the apparatus and fill the chambers with 1X TBE buffer. It is recommended to perform a prerun for about 10 min at 5 V/cm.
- Prepare the oligonucleotide samples by mixing 1 µL of sample with 5 µL of 5X gel loading buffer. Load the samples carefully into the wells without introducing any air bubbles.
- Large gels may be run ~70 V for 14-16 h or 125-150 V for 2-4 h or until the dye front approaches the bottom of the gel. Ensure that there is no excessive heating. Alternatively, the samples may be run at a higher voltage in a cold room.
- Pry open the glass plates with a spatula, separate the upper glass plate. Stain the gel while still attached to the lower glass plate with 0.5 µG/mL ethidium bromide solution for 5 – 10 min.
- Soak the gel on the glass plate in distilled water for 10 – 30 min to remove excess stain and lower the background staining.
- Wrap the gel and plate in plastic wrap, invert onto UV transilluminator and acquire the image of the gel.
- The samples will appear as bright bands.
- The desired bands may be cut with a fine scalpel and processed appropriately to recover the DNA.
If 35S or 33P nucleotides are incorporated in the oligonucleotides, follow the procedure below:
- Soak the gel in 10% acetic acid for 15 min.
- Pat the gel dry and wrap with plastic wrap.
- Dry the gel in vacuum drier at 80 °C for 20 min or longer.
- Remove the plastic wrap and expose the gel to X-ray film in dark.
Figure 3.Vertical Electrophoresis System
Reference
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