Exp 4.

Agarose Gel Electrophoresis

&
Exp 5. Nucleic Acid Quantification
 Introduction: Agarose

• The monomeric unit of Agarose is a disaccharide.

• Agarose is a linear polymer of alternating D & L galactose

residues joined by alpha (1→3) and beta (1→ 4) glycosidic
linkage.

• Gelation of agarose results in a three-dimensional mesh of channels whose diameters range from 50 nm to >200 nm.

• The gelling properties are attributed to inter- and intramolecular hydrogen bonding within and between the long agarose chains.

• The initial concentration of agarose controls the pore size in the gel; large pore sizes are formed at low concentrations, and
smaller pore sizes are formed at higher concentrations.
 Introduction: Agarose

• It is used to separate, and purify the DNA fragments.

• By using agarose gel, 50 bp to several megabases (20-30 kb) can be separated.

• Location of DNA is identified by using EtBr or SYBR gold.

• 20 picograms of DNA can be detected by directly examining the gel in UV light.

• Agarose gels have lower resolving power but have excellent separation range (compared to PAGE).

• Agarose is not homogeneous. Lower-grade agarose may be contaminated with other polysaccharides, salts, and proteins.
Introduction: Agarose
Agarose gel electrophoresis
 Ethidium Bromide (EtBr)
• It is used to visualize the DNA bands.

• It is a Phenanthridine compound discovered in the late 1950s and initially used as a trypanocidal agent, especially in veterinary.

• Sharp et al., 1973 used EtBr in DNA staining due to its fluorescence property after intercalation.

• One EtBr molecule intercalates per 2.5 bp independent of the base composition of the DNA. EtBr dye lies perpendicular to the
helical axis, making Vander Waal contact with base pairs.

• UV radiation at 254 nm is absorbed by DNA and transmitted to the dye, and the dye itself absorbs radiation at 302 nm and 366
nm. The fraction of energy is remitted at 590 nm (red-orange region).

• EtBr also binds to highly variable secondary structures of RNA.

• 10 mg/ml stock concentration is prepared, and 0.5 μg/ml standard final

concentration of EtBr is used in Agarose gel preparation.

• It can detect 10 ng of DNA in the gel.

• EtBr is not used in Polyacrylamide gel preparation as it hinders the polymerization

of acrylamide.
 CAUTION
Use great care when weighing out powdered ethidium bromide. Ethidium bromide is a mutagen
and probable carcinogen. Ethidium bromide is toxic. Wear gloves and a face mask when working
with ethidium bromide in powdered form. Wipe the area with a damp cloth after completing the
work with ethidium bromide powder. A safer alternative is to purchase ethidium bromide in
solution. This eliminates the hazards of working with the powdered form. Always wear gloves
when working with ethidium bromide and ethidium-stained gels.

The Myth of Ethidium Bromide

https://www.science.org/content/blog-post/myth-ethidium-bromide
 Gel loading buffer

• It increases the sample's density (addition of Sucrose/ Glycerol); 6X is prepared and stored.
• Add color to the sample. Bromophenol blue runs twofold faster than Xylene cyanol.

• Despite having a more considerable molecular weight, Bromophenol blue (670 g/mol, 400-500
Bromophenol blue
bp) runs faster than Xylene cyanol (539 g/mol, 3000 -4000 bp size) because of negative charge.

• Orange G is another dye that runs 50 bp faster than these.

• Their running also depends upon the running buffer used.

• Composition:

Xylene cyanol

6X Gel Loading Buffer

0.25% Bromophenol Blue

0.25% Xylene Cyanol
40% (w/v) Sucrose in Water
The rate of migration of DNA through agarose gel depends upon:

• The molecular size of DNA: Larger molecules move slowly due to frictional drag.

• The concentration of Agarose: There are different rates in different agarose percentages.

• The conformation of DNA: A super-helical form of plasmid DNA moves faster than
other forms (nicked circular or linear).

• The presence of EtBr in the gel: Intercalation retards the speed by 15%.

• Electrophoresis buffer (Tank buffer): TAE (Tris Acetate EDTA), TBE (Tris Borate
EDTA), and TPE (Tris Phosphate EDTA) are generally used. TBE and TPE are adequate,
with high buffering capacity, but costly. TAE is economical but has low buffering
capacity.

• Applied voltage: The migration rate is proportional to the applied voltage. More than 2 kb DNA should not be run more than
5-8 V/cm

• Type of Agarose: The agarose contains charged groups, e.g. sulfate, which can retard the movement of DNA.
 The average molecular weight of a base
pair (depending on the sequence) is roughly
around 650 Daltons (g/mol) or 650 g/mol.

Disclaimer: I do not own this content. All credits go to its rightful owner. It has been designed for educational purpose only.
Nucleic acid quantification
• Nucleic acids are quantified in terms of Concentration and Purity for
their downstream processing.

• For purity, the ratio of absorbance value at 260 nm and 280 nm is

considered.

• The absorption maximum of DNA and RNA nitrogenous bases is 260 nm.

• At 280 nm, protein aromatic amino acids absorb more.

• Absorbance at 230 nm indicates contamination from phenolate ions or

organic compounds, and absorbance at 320 nm indicates contamination
from polysaccharides or particulate matter.

• A ratio of ~1.8 (between 1.6- 1.8) is generally accepted as

“Pure” for DNA.

• A ratio of ~2.0 (between 1.8 to 2.0) is generally accepted as

“Pure” for RNA.
• If the ratio is appreciably lower in either case, it indicates the presence of proteins, phenol, or other contaminants that absorb
strongly at or near 280 nm.

• In addition, the 260/230 ratio is also calculated. This ratio is used as a secondary measure.

• Expected 260/230 values are commonly in the range of 2.0-2.2.

• If the ratio is appreciably lower than expected, it may indicate the presence of contaminants that absorb at 230 nm.

• Absorbance at 320 nm is considered as background reading, and it is subtracted from the peak. Contaminations with
chaotropic salts can also lead to increased light scatter. Measuring and correcting the reading at 320 nm removes any
interference from the light scatter, from the cuvette, polysaccharides, particulate matter, etc.

• All five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios. The following represent the
260/280 ratios estimated for each nucleotide if measured independently:

Guanine: 1.15, Adenine: 4.50, Cytosine: 1.51, Thymine: 1.47, Uracil: 4.00
• Concentration: The Absorbance at 260 nm is used as a quantity measure for nucleic acids.

1 A260 unit for dsDNA corresponds to 50 microgram/ ml

1 A260unit for ssDNA corresponds to 33 microgram/ ml
1 A260 unit for ssRNA corresponds to 40 microgram/ ml
1 A260 unit for ssOligo corresponds to 20 microgram/ ml
Laboratory exercise: Nucleic acid quantification

Objective: Analyse quality (purity) and quantity (amount) of nucleic acid

Procedure:

1. Take 1 µl of Nucleic acid sample and add 999 µl of Autoclaved double distilled water in fresh
tube.

2. Switch on the spectrophotometer (20 min before) and set zero by filling both cuvette with
autoclaved distilled water.

3. Take out one cuvette and fill diluted sample.

4. Now take OD of your sample at different wavelength as mentioned below and record.

5. Now calculate the concentration and purity according to the formula given.

Note: Take OD at 260 nm, 280 nm, 230 nm, 320 nm

OD value should range between 0.1 to 1.0

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50 µg/ml

(Note: 40 µg/ml in case of RNA)

Nucleic acid purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320
reading)
 Makeup labs (Exp 1-5): 28th and 29th Jan

Disclaimer: I do not own this content. All credits go to its rightful owner. It has been designed for educational purpose only.