BIO103 Lab

(1 credit hour)
Spring-2021

Dept. of Biochemistry & Microbiology

Experiment 3 and 4
 Experiment 3
Biuret test for determining the presence of milk proteins

•  The most abundant and diverse molecules found in living cells are
proteins.

•  One of the macromolecule that composed of one or more polypeptide

chains, each with a characteristic sequence of amino acids linked by
peptide bonds.

•  The building blocks of proteins are amino acids (AA’s).

•  There are 20 different AA’s that make up proteins.

•  The different amino acids are similar in structure, have different side
chain,
•  The center of the molecule is the alpha carbon that is connected to an amino
group, a carboxyl group, a hydrogen atom and the R group (the side chain). Peptide bond
 Functions of Proteins

•  Proteins main function is to build, maintain and repair all our body tissues.

•  Protein can also be used as energy source by body.

•  Some proteins have a protective or defensive function.

•  Proteins serve as the carrier and transporters of essential substances.

•  Types of proteins: Structural proteins & Regulatory proteins.

□  Structural proteins i.e., collagen or elastin

□  Regulatory proteins i.e., enzymes

 Milk Protein

•  Milk contains 3.3% total protein.

•  Contain all 9 essential amino acids required by humans.

•  There are two major categories of milk proteins,

□  casein and

□  whey proteins.

•  Milk proteins are synthesized in the mammary gland.

 Principle

•  Reaction of Biuret reagent with peptide bonds forming

chelate complex.

•  Cu (II) ion containing biuret reagents, interact with a

compound that contains at least four peptide bonds,
resulting in the formation of violet/purple colored complex.

•  A color change from blue to violet indicates that proteins

are present.

•  The greater the concentration of peptide bonds, the greater

Reaction of Biuret reagent with
the color intensity. peptide bonds forming chelate
complex.
 Principle (cont.)
•  If the concentration of peptide bonds is low, it results the color change is from
the blue to pink.

•  Single amino acids (no peptide bonds present) and dipeptide (only 1 peptide
bond present) which will give a negative result.

•  Peptides with at least two peptide bonds are required for the formation of the
chelate complex.
 EXPERIMENT

•  Apparatus
•  Procedure

□  Glassware
1. Add 2 ml of the milk sample to a clean and dry test
□  Test tubes
tube.
□  Test tube holder
2. Add 2 ml of biuret regent.
□  Dropper
3. Repeat the steps above with distilled water to
prepare a negative control.
•  Reagents
4. Shake well and allow the mixture to stand for 5
□Biuret reagents minutes.
□  Test sample (liquid milk) 5. Observe any color change.
□  Distilled water
 Experiment 4
Isolation of plant genomic DNA from spinach

•  Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for
the development and function of living things.

•  The structure of DNA is called a double helix, which looks like a twisted staircase.

•  The main role of DNA in the cell is the long term storage of information's.

•  There are four different bases:

□adenine (A), cytosine (C), guanine (G), or thymine (T).

•  The two strands are held together by hydrogen bonds between the bases, with adenine
forming a base pair with thymine, and cytosine forming a base pair with guanine.
 DNA Extraction

•  DNA can be isolated from living tissues e.g., spinach, banana, onion.

•  DNA isolated from the nucleus, for which cell wall and plasma membrane must be broken
down by mechanical forces, in the presence of salt and detergent.

•  Isolation of DNA from plant material poses special challenges, i.e., several plant
metabolites have chemical properties similar to DNA.

•  Young and healthy tissues are recommended to use for DNA extraction

□  Young tissues contains more cells than older tissue.

□  Young tissue of the same weight contains fewer metabolites.

 Principle
•  The process of isolating nucleic acid is a crude extraction method.

•  By using salt and detergent solution causes the cell membrane to break down or
lyse and emulsifies the lipids and proteins of the cell.

•  After incubation period, the crude mixture is filtered to get homogenized solution.

•  Pouring of this solution into petri dish and glass tube containing 95% ethyl alcohol
or ethanol, causes the precipitation of DNA in time.

•  After some moment, it appears as thick, stringy, white or slight greening mass.

•  As DNA is not soluble in alcohol, can be isolated as they clamp together and
become visible to the naked eye.
 EXPERIMENT

•  Materials •  Procedure

□  Measuring cylinder, beakers

Chop and grind sample
□  Mortal and pestle

□  Light colored dish washing liquid

Add liquid with detergent and salt
□  Ethanol 95% solution

□  Large spoon for mixing

□  Petri dishes Incubate at 56°C for 10 minutes

□  Strainer/ funnel

□  Salt Filtration
□  Filter cloth or paper

Add 95% ethanol and visualize DNA

Thank
you!