AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones .
- The basic principle of separation for all electrophoresis is the movement of a charged molecule in a medium subjected to an electric field.
- V is the velocity of the molecule subjected to electrophoresis.
- E is the electrical field in volts/cm.
- q is the net charge on the molecule.
- f is the frictional coefficient.
- The impact of f depends on the mass and shape of the molecule.
- Agarose gel electrophoresis used to analyze and quantitative nucleic acid.
- The Agarose for Agarose gel electrophoresis is purified from agar.
- Agarose is a seaweed extract (red algae agar) and is a long polymer of D and L galactose and derivatives in a linear polymer bonded by two different glycosidic bonds.
- Agarose has an average MW of 12,000 and contains about 35-40 agarobiose units
- Once hydrated and formed into a gel, the carbohydrate will form helical fibers and aggregates creating channels of 50 to more than 200 nm in diameter.
- DNA and RNA molecules migrate by ‘reptation’ (snaking through this matrix).
- polymer made up of repeating units of 1,3 –linked ß D galactopyranose and 1, 4 linked 3,6 anhydro a L galactopyranose [ P-D –gal (1-4)-3,6 anhydro – a L Gal (1-3) ]n.
- Agarose in solution exist as left handed double helices.
- About 7 to 11 such helices form bundles which extend as long rods and appear to intertwine with one another, further strengthening the frame work of the gel.
- The cross links are held together by hydrogen and hydrophobic bonds.
- Because of large pore size even at low concentration, Agarose gels are widely used for separation of DNA and RNA.
TYPES OF AGAROSE
- STANDARD AGAROSE has a high strength and high melting temperature.
- Melting temp: 85-95oC gelling temp: 34-42oC.
- “LOW MELTING TEMPERATURE AGAROSE” melt at 63-65oC and gel at 25-35oC.
- A quality agarose will have a low electroendo-osmosis (EEO). That is the tendency of negatively charged sulfates in agarose to induce positive ions in the buffer causing the DNA to migrate in the opposite direction.
- MOLECULAR BIOLOGY AGAROSE: This is a general-purpose agarose that has a high exclusion limit.
- This type of agarose has high gel strength and is easy to handle at low percentages.
- LOW-MELT AGAROSE: The main use of low-melt agarose is for preparative electrophoresis.
- It is ideal for in-gel applications such as ligation, PCR, restriction enzyme digestion, transformation, and sequencing.
- Other applications include pulsed field electrophoresis of megabase DNA and embedding chromosomes.This agarose has a gelation temperature of 26°C .
- PCR AGAROSE: This high-strength agarose forms gels that are easy to handle and remain flexible even at high gel percentages, reducing the risk of cracking or breaking.
- Gelation temperature is 40°C.
- This agarose has excellent sieving properties and the highest gel strength of all the agaroses.
- PCR LOW-MELT AGAROSE has a high sieving capacity. It is ideal for preparative electrophoresis and in gel applications such as restriction enzyme digests, ligation, and transformation.
- PCR agaroses are recommended for DNA fragments <1,000 bp.
- There are a few basic buffers for running DNA gels. TAE, TBE, TPE and Borate buffers (not the same as a TBE buffer). Each has a different use.
- TAE is best used if recovering DNA from gel slice, while TBE is better for smaller (< 1 kB) DNA strands.
TAE Buffer (Tris/Acetate/EDTA)
TBE Buffer (Tris/Borate/EDTA)
- Use when DNA is to be recovered.
- Use for larger than 12kb DNA .
- Low ionic strength.
- Low buffering capacity- may need to recirculate for extended runs.
- Used for less than 1 kb DNA –provides tighter bands with higher % gels.
- Decreased DNA mobility.
- High ionic strength.
- High buffering capacity.
- Not best if recovering DNA after run.
- High buffering capacity.
- Will work for recovering DNA after run.
- Good for long runs.
- Used for analysis of single stranded DNA.
- Will interfere with phosphate sensitive reactions of recovered DNA.
Na BORATE BUFFER
- Used for high voltages providing faster runs.
- Limited resolutions.
- Best for quick analytical gels of purified DNA or restriction digests.
- STAINING WITH ETHIDIUM BROMIDE (EtBr) is a rapid, sensitive, and highly reliable method for visualizing DNA in gels.
- The stained gel is illuminated from below (‘transillumination’) with short- or medium wavelength of UV light causing the EtBr, bound to DNA, to fluoresce brightly.
- As a molecule that binds DNA, however, EtBr is a mutagen and likely carcinogen.
- EtBr should be handled with appropriate caution, and its use restricted to limited areas.
- SYBR Safe DNA Stain [Safe Alternative] SYBR Safe is a cyan based, non-mutagenic dye (no observed in an acute oral toxicity study in rats) form of SYBR Green dye.
- The dye absorbs in the blue range, fluoresces only when complexes with DNA and then emits in the green (lambda max 520 nm).
- The dye is purchased as a highly concentrated stock (often 10,000X). Add to cooling agarose gel before pouring into a casting stand.
- SYBR Safe stain can use blue light for visualization avoiding UV damage to DNA .
PREPARATIONS OF ETHIDIUM BROMIDE (STOCK SOLUTION)
- Weigh 10 mg ethidium bromide into a sterile tube and dissolve in 10 ml sterile distilled water. The stock is stored at 4 degree Celsius.
PREPARATION OF SAMPLE LOADING DYE GLYCEROL & BROMOPHENOL BLUE (6X)
- 3ml glycerol (30%), 25mg bromophenol blue (0.25%) dH2O to 10mL .
- Dissolve the Agarose by placing the flasks in boiling water both cool to Luke warm.
- Cover the sides of a tray using cello-tape and place the comb about 1 cm from the top of the tray.
- Pour the Agarose with out making any bubbles, cool it for 20 mins and take off the combs and uncover the cello-tapes .
- The DNA sample (100 to 200 ng) is mixed with the loading dye (for 5 µl of DNA sample 1µof 6x dye is used) and loaded in to the well carefully, using a pipette or capillary tube.
- Once the sample is loaded in to the well, the cathode (Black negative terminal) is connected towards the top end of the gel and the anode (Red positive terminal is connected towards the bottom end of the gel.
- The maximum volume that can be loaded on to a well formed from a 1.5 mm thickness tooth of the comb is 30 µl.
- The electrophoresis is started by switching on the D. C. Power.
- The gel is run at 5 v/cm.
- As the bromophenol blue(the tracking dye) has moved 1 cm above the bottom end, the current is switched off, the power supply is disconnected .
- The gel along with the platform is stained in the plastic tray containing 0.5 µg/ml ethidium bromide in the sterile distilled water( use gloves when handling ethidium bromide).
- After about 30-45 min, the platform and the gel is rinsed with distilled water and by keeping the platform in a slanting position, the gel is gently pushed onto the UV Transilluminator.
- UV light is switched on and the DNA bands are seen and Photographed at f 5.6 for 10 seconds with an orange filter.
- Estimation of the size of DNA molecules.
- Analysis of products of a polymerase chain reaction (PCR).
- Separation of DNA fragments for extraction and purification.
- In the field of forensic science, molecular biology.
- The main benefit of agarose gel technique is that it can be easily processed.
- Agarose gel does not denature the DNA samples and they stay in their own form.
- There is also a disadvantage of agarose gel electrophoresis that it may melt when the electric current is passed through it.