DNA STRUCTURE

Lesson 2
Lecturer: Niña Betina Marie M. Igaña, MPH(c)

OBJECTIVES:
1. DNA STRUCTURE
1.1. Nucleotide
1.2. Chargaff’s Rule
1.3. Central Dogma of Biology
1.4. The Genetic Code
2. DNA EXTRACTION
2.1. Extracting DNA from living organisms
2.2. DNA Extraction Experiment (homework)
2.3. Know the Appearance of Extracted DNA

THE CHEMICAL COMPOSITION

● DNA and RNA are polynucleotides, meaning they are made up of repeating units of nucleotide
● The sugar component of the genetic material is a five-carbon molecule or called a pentose
● For DNA: the sugar is deoxyribose
● For RNA: the sugar is a ribose

NUCLEOTIDES
● Nucleotides are the monomer units of building blocks of nucleic acids. It is a compound consisting of a
nucleoside linked to a phosphate group.
● They form a part of many coenzymes. Nucleotides form the basic structural unit of nucleic acids such
as Deoxyribonucleic Acid or DNA, and Ribonucleic acid or RNA.
● The universal currency of energy, namely ATP, is a nucleotide derivative.
○ Nucleotides are concerned with the storage and transfer of genetic information
● Serve as donors of phosphoryl groups (eg. ATP or GTP) of sugars (eg. UDP- or GDP sugars), or of
lipid (eg. CDP-acylglycerol)
○ There are coding and non-coding regions found on DNA.
○ For the coding regions, these code for genes such as proteins
○ For the non-coding regions, can be either for DNA junk or to help regulate protein synthesis.

NITROGENOUS BASES
● These are ring structures or aromatics that contain nitrogen as well as carbon in their rings thus
referred to as heterocyclic.
● The Nitrogen bases can either be:
○ PURINES: Adenine (A), Guanine (G)
○ PYRIMINDES: Cytosine (C ), Thymine (T), Uracil (U)
● Purines and Pyrimidines are cyclic compounds whose rings contain both carbon and other elements
(hetero atoms)

PURINES PYRIMIDINE

Adenine, Guanine Cytosine, Thymine, Uracil

Double ring structure Single ring structure

-the purine ring and five-membered imidazole
ring composes the purines

Chemical Formula: C5H4N4 Chemical Formula: C4H4N2

 The DNA molecule has A,G,C and T,
While RNA also has A,G and C but T was replaced with U or Uracil

● Purines and Pyrimidines are the nitrogen bases that hold DNA strands together through hydrogen
bonds
○ The purines in DNA are adenine and Guanine, same as in RNA
○ The pyrimidines in DNA are cytosine and thymine, but in RNA they are cytosine and Uracil.

● A purine contains a pyrimidine ring fused with an imidazole ring or a five-member

ring with two non-adjacent nitrogen atoms)
● This two-ringed structure has nine atoms forming the ring: 5 carbon atoms and 4
nitrogen atoms
● Different purines are distinguished by the atoms or functional groups attached to the
PURINE rings
● Purines serve much the same function as pyrimidines in organisms
○ Naming these functions are cell signaling, energy storage, and enzyme
regulation
○ The molecules are used to make starch and proteins
○ So purines are abundant in meat, fish,beans, peas, and grains

● A pyrimidine is an organic ring consisting of six atoms: 4 carbon atoms and 2

nitrogen atoms
PYRIMIDINE ● The nitrogen atoms are placed in the 1 and 3 positions around the ring
● Pyrimidines function in DNA and RNA, same function in purines:
○ cell signaling, energy storage (as phosphates), enzyme regulation and to
make protein and starch.

● These are the structures of purines and

pyrimidines
● Base + Sugar + Phosphate= Nucleotide
● Purines is a heterocyclic aromatic organic
compound consisting of a pyrimidine ring
fused to an imidazole ring, which means it
has 2 rings
● Pyrimidine is a heterocylic aromatic
organic compound, similar to purine,
containing 2 nitrogen atoms at positions 1
and 3 of the member ring

DNA STRUCTURE
● DNA stands for Deoxyribonucleic Acid
○ D from the name of the sugar
○ N and A from Nucleic Acid
● DNA contains the information that determines inherited characteristics
● It has the code for making proteins
● DNA is found in the nucleus of eukaryotic cells and in the cytosol of prokaryotes if we take a closer look
at the chromatin inside the nucleus
○ Cells require some form of instructions to be able to function properly. They need guidelines,
rules, and codes for making materials in the cell, and that code is the DNA

THE STRUCTURE OF DNA

● A nitrogen base covalently binds with a sugar to form nucleoside
● The bond formed is N-glycosidic bond
● The nucleoside in turn binds covalently to make a phosphate group to form a nucleotide
○ The covalent joining the nucleotides make up the polynucleotide
● As a result of the joining, the phosphate group forms two covalent bonds with two adjacent sugar
residues
● One of the PO4 groups (phosphate groups) is bound to carbon number 5 of one sugar molecule and
the other to carbon number 3 of the adjacent sugar molecule. This is known as the phosphodiester
linkages or bridges.
● Video time stamp (09:00)

● Let’s talk further about the structure of the DNA. The DNA has repeating sub units and those units are
called monomers or nucleotides.
● The nucleotide has three main parts, a phosphate group, a sugar, and a nitrogen base.

● In the DNA, the name of the sugar is deoxyribose, which is part of the DNA’s name, and there are for
nitrogen bases, namely: Adenine, Guanine, Cytosine, and Thymine

● Two of the bases are purines, which have two ring structures: Adenine and Guanine
 ● Two of the bases are pyrimidines, which have one ring structure: Cytosine and Thymine. Pyrimidines
are the bases with a “y’ in their name, just like cytosine and thymine and pyrimidine itself.
● Appearing always in pairs with a pyrimidine and the slanted shape of DNA molecule causes it to form a
spiral or helix.
● Along the sides of the molecule, it is a backbone made up of alternating sugar and phosphate
molecules. While on the inside are nitrogen bases.
● Adenine and Thymine form hydrogen bonds together while Cytosine and Guanine form hydrogen
bonds together.

To help remember: THE DNA STRUCTURE

● A & T use straight lines while C & G use curved lines
● A & T have 2 hydrogen bonds (AT 2)
● C & G have 3 hydrogen bonds (CG 3)

Strands of DNA are said to be complementary to one another because A

will always be with T and C will always be with G and will never
interchange.

● Sugars and Phosphates make the

outside of the molecule.
● Deoxyribose is a pentagon shape
● Phosphate groups are in between the
sugars
● Nucleotides with 2 hydrogen bonds are
AT
● Nucleotides with 3 hydrogen bonds are
CG
● Pyrimidines have 1 ring (CT)
● Purines have 2 rings (AG)

DNA VS. RNA

DNA RNA

Stands for: Deoxyribonucleic Acid Ribonucleic Acid

Difference: 1. Found in the Nucleus 1. Found in the nucleus and cytoplasm

2. Sugar is deoxyribose 2. Sugar is ribose
3. Bases are A, T, C, G 3. Bases are A, U, C, G

Bases & DNA is a long polymer with a deoxyribose RNA is a polymer with a ribose & phosphate
Sugars: & phosphate backbone backbone

Job/Role: Medium of long-term storage and Transfer the genetic code needed for the
transmission of genetic information creation of proteins from the nucleus to the
ribosome

Description: A nucleic acid that contains the genetic Single-stranded chain of alternating
instructions used in the development & phosphate & ribose units with the bases A, G,
functioning of all known living organisms C, U bonded to the ribose.

Predominant Typically double-stranded molecule with a A single-stranded molecule in most of its

structure: long chain of nucleotides biological roles

Pairing of A-T (Adenine & Thymine) A-U (Adenine & Uracil)

bases: G-C (Guanine & Cytosine) G-C (Guanine & Cytosil)

Stability: Deoxyribose sugar in DNA is less Ribose sugar is more reactive.

reactive.
 Stable in Alkaline solutions. It is stable
because of the carbon-hydrogen
bonds.DNA has smaller grooves where the
damaging enzyme can attach which makes
it harder for the enzymes to attack DNA.
Not stable in Alkaline solutions. The ribose
sugar is more reactive thus it is not stable in
alkaline solution. It is because of the c-o
bond. RNA has larger grooves which makes it
easier to be attacked by the enzymes.

Unique The helix geometry of the DNA is of The helix geometry of RNA is of A-form.
features: B-form.

DNA is completely protected by the body. RNA strands are continually made, broken
Because the body destroys enzymes that down and reused.
cleave DNA.

Can be damaged by exposure to More resistant to damage by Ultraviolet (UV)

Ultraviolet (UV) rays. rays.

CHARGAFF’S RULE

● Erwin Chargaff founded in 1940 that DNA differs in different

species. Each species has a different ratio of bases.
● This is the Chargaff’s rule of molar equivalence between the
purines and pyrimidines in DNA structure. DNA has an equal
number of adenine and thymine residues and an equal number of
guanine and cytosine residues.
● The double helical structure of DNA derives its strength from
Chargaff’s rule. There are two possible helical forms of DNA as
mirror images of each other.
● Single stranded DNA & RNAs which are usually single stranded do not obey the rule.
● After the development of a method for the precise chemical characterization of nucleic acids
● Chargaff, in 1950, observed using current language that in any double-stranded DNA segment, the
Adenine and Thymine frequencies are equal, and so are the frequencies of Cytosine and Guanine
(Chargaff, 1950). This observation is known as Chargaff’s first parity rule.
● Chargaff also perceived that the parity rule approximately holds in the single-stranded DNA
segment. This last rule is known as Chargaff’s second parity rule (CSPR), and although it is not well
understood, it has been confirmed in several organisms (Mitchell & Bride, 2006).
● DNA composition vary from specie to specie
○ Made DNA a more credible genetic material than protein
● The number of Adenine & Thymine are equal. The number of Cytosine and Guanine are equal.
● Thanks to the Chargaff’s rule, Watson and Crick got to defend the double helix structure of DNA. This
proved the composition of the DNA through the base sparing.
● Watson and Crick were acquainted with this rule and used it to support their famous DNA double helix
structure model (Watson & Crick, 1953).

CENTRAL DOGMA OF BIOLOGY

● Dogma means truth.

● It describes the flow of genetic information in all of life.
● Process by which the instructions in DNA are converted into a functional product.
● It was first proposed in 1958 by Francis Crick, discoverer of the structure of DNA.
● DNA as the genetic material stores all the information for all the traits of an individual in the
nucleotide base sequence.
● However, traits are expressed through the action of proteins.
● But how does DNA control the synthesis of a protein?
 CENTRAL DOGMA OF MOLECULAR BIOLOGY
The central dogma of molecular biology explains the flow of genetic information from DNA to RNA to make a functional product
called the protein.

This is the cell, the basic unit of all living tissue. So,
in most human cells there is a structure called the
nucleus. The nucleus contains the genome.

In humans, the genome is split between 23 pairs of

chromosomes.

So, each chromosome contains a long strand of

DNA, tightly packaged around proteins called
histones.

So, within the DNA are sections called genes. These

genes contain the instructions for making proteins.
When a gene is switched on, an enzyme called RNA
polymerase attaches to the start of the gene. It
moves along the DNA, making a strand of
messenger RNA out of free bases in the nucleus.

The DNA code determines the order in which the

free bases are added to the messenger RNA.

This process is what we know as transcription.

Before the messenger RNA can be used as a

template for the production of proteins, it needs to be
processed. This involves removing and adding
sections of RNA. The messenger RNA then moves
out of the nucleus into the cytoplasm.

Protein factories in the cytoplasm called the

ribosomes bind to the messenger RNA. The
ribosome then reads the code in the messenger
RNA to produce a chain made up of amino acids.
 As you all know, there are twenty different types of
amino acids. So, transfer RNA molecules carry the
amino acids to form the ribosomes.

The messenger RNA is read three bases at the time.

As each triplet is read, a transfer RNA delivers the
corresponding amino acid.

This is then added to a growing chain of amino

acids.

Once the last amino acid has been added, the chain
folds into a complex 3D shape to form a protein.

Genetic information travels from the DNA to the RNA and all species contain DNA that contains our genetic
information.
 REPLICATION TRANSCRIPTION TRANSLATION

DNA is capable of replication In transcription, RNA polymerase In translation, the mRNA is read
using DNA polymerase. So, this uses DNA as a template to make as a template by the rRNA and
shows when our hair is growing, RNA. proteins and ribosomes which also
nails are growing, bacteria is uses tRNA to make protein out of
spreading within our bodies. This mRNA ● Messenger RNA. amino acids. Proteins go on to
happens because our cell is ● Contains all the perform their cellular functions,
replicating. DNA contains genetic DNA information. such as replicating,
information, then it is transferred phosphorylating, signaling aspect
to the RNA through transcription. rRNA ● Ribosomal RNA. of the cell.
● Complexes with
proteins that make
up the cellular
organelle called
the ribosome.

tRNA ● Transfer RNA.

● Carries amino
acids which works
with the rRNA to
enable the
process of
translation

SPECIAL TRANSFERS
The Extensions of the Central Dogma

REVERSE TRANSCRIPTION RNA REPLICATION

RETROVIRUSES RNA REPLICASE

● These are viruses with RNA as the genetic
material.
● These contain a unique enzyme called
reverse transcriptase.
○ This enzyme can catalyze the
enzymatic synthesis of a DNA
complementary to the viral RNA.
○ Examples: Human Immunodeficiency
Virus (HIV) and AIDS
● RNA-containing viruses when present in their
host cell (Ex: E.coli) bring about the synthesis
of a set of enzymes.
○ Because of the presence of these
enzymes the duplication of the virus
RNA is made possible.

GENETIC CODE

● Four basic requirements for a biomolecule to qualify as a genetic material.

○ The amount of information stored in the genetic material should account for virtually all
expressible traits, which is unimaginably large. The DNA could meet this by equally
unimaginable variation in base sequences.
● The genetic code has the:
a. Ability to store large amounts of genetic information.
b. Ability to transfer the information to daughter cells.
c. Physical and chemical stability of the cell.
d. Mutability.
● Ultimately the base sequences are responsible for “dictating” the amino acid sequences of proteins
which, in turn, directly and indirectly express the organism’s trait.
● The DNA is a very stable biomolecule mainly due to the three factors inherent in its structure
I. Sugar-phosphate backbone
 - It is extremely stable under all conditions like extreme temperatures and pH because
the sugar phosphate backbone.
II. Base-stacking
- Which refers to the tendency of the hydrophobic end basis to pile on top of the other.
- Also contributes to the stability of the genes.
III. Hydrogen bonds
- They are weak themselves. They can add tremendous stability when found between
millions of base pairs of a whole DNA structure.
● RNA is not an ideal genetic material because its structure, though stable, loses much of the base
stacking and almost all of the hydrogen bonding.

WHAT IS GENETIC CODE?

● Genetic code is a dictionary that corresponds with sequence of nucleotides and sequence of amino
acids.
● Genetic code is a set of rules by which information encoded in genetic material (DNA or RNA
sequences) is translated into proteins by living cells.
● Term given by “Goerge Gamow” – when i searched the name up it was spelled “George”.
● The letters A,G,T and C correspond to the nucleotides found in DNA. They are organized into
Codons.
● The collection of codons is called Genetic code.
● For 20 amino acids there should be 20 codons.
● Each codon should have 3 nucleotides to impart specificity to each of the amino acid for a specific
codon.
● 1 nucleotide – 4 combinations
● 2 nucleotide – 16 combinations
● 3 nucleotide – 64 combinations ( most suited for 20 amino acids )

GENETIC CODE

● The DNA act as a genetic material that

encodes all the information required to
encode a protein.

● The genetic code is a particular sequence of

nucleotide that tells which amino acids are to
be linked together to form a protein.
● The genetic code consists of 64 triplets of
Nucleotides that codes for 20 amino acids.
● Each of these triplets are known as “Codon”.

● Now, as there are 64 codons and only 24

amino acids, a single amino acid can be
coded by more than one codon – This
property of genetic code is known as
Degeneracy.
● For example, amino acid Proline can be
coded by CCU, CCC, CCA, and CCG.

● However, a single codon will all be scored for

a single amino acid.
● For example, CCU, all the scores for Proline.
AUG always codes for Methionine.
○ This property of genetic code is called
“Non ambiguity”. Thus, one codon
can not specify more than one amino
acid.

● The genetic code is non overlapping. Which

means, after reading one triplet, the reading
frame shift over the other three nucleotides.
● One base can not participate in the formation
of more than one codon.
● The genetic code is of continuous translation.
● It is “Coma less” , which means there is not
extra nucleotide between the codons.
○ The gene is transcribed and
translated continuously from a fixed
starting point to a fixed stop point.

● The genetic code has a start and stop codon

○ AUG as usually a start codon that
codes for Methionine. And the stop
codon, are UAA, UAG, and UGA – a
stop codon is also known as
Nonsense codon as they do not code
for any amino acid.
 ● The genetic code is nearly universal. Which
means, it is the same in humans, bacteria,
plants, and amphibians.
● However, there are some exceptions in
mitochondrial genome.
● The genetic code is also universal in a way
that AUG is the codon for Methionine in the
mitochondria, the same code which is AUG,
codes for Isoleucine in the cytoplasm.
● With some exceptions noted, the genetic
code is universal.

POLARITY ● Lastly, the code has polarity.

● The code has a definite direction for reading
● Definite direction for reading of messages – which is referred to as the
○ UUG -> LEUCINE “polarity”.
○ GUU -> VALINE ● Reading of messages from left to right and
right to left will specify for different amino
acids – which means, you can not
interchange them.
● For example. UUG stands for leucine. And
from right to left, it is GUU which stands for
Valine.

TYPE OF CODON ANTICODON

● Sense Codons ● The base sequence of t RNA which pairs with

○ Codons that codes for amino acids, codon of mRNA during translation is called
thus it is called the “sense codons”. anticodon.
● Signal Codons
○ Codons that codes for signal during
protein synthesis.
○ Examples of signal codons are AUG,
UAA, UAG, and UGA.
○ Two types of signal codons:
■ Start Codons (initiating
codon)
● The start codon, AUG
specifically, is the
initiation codon. It
codes for the first
amino acid in all
proteins.
● At the starting point it
codes for Methionine in
Eukaryotes and
formylmethionine in
prokaryotes.
■ Stop Codons (terminating
codon)
● The stop codons UAA,
UAG, and UGA are
termination codons or
nonsense codons as
mentioned earlier.
● These are often
referred to as Amber,
Ochre, and Opal
codons.
 DIFFERENCE BETWEEN CODON AND ANTICODON

● Codon could be present in both DNA & RNA, but anticodon is always present in RNA & never in DNA
● Codons are written in 5 to 3 direction whereas anticodons are usually written in 3 to 5 direction.
● Anticodon of some tRNA molecules have to pair with more than one codon.
● Codons are sequentially arranged in nucleic acid strand while anticodons are discreetly present in
cells with amino acids attached or not.
● Codon defines which anticodon should come next with an amino acid to create the protein strand.
● Anticodon helps in bringing a particular amino acid at its proper position during translation.

CLINICAL SIGNIFICANCE

● Mutation can be well explained using the genetic code.

● A) Point Mutations
1.) Silent
2.) Missense
3.) Nonsense
4.) Frame shift mutations

● These are mutations in DNA that do not have an observable

effect on the organism’s phenotype.
● Single nucleotide change-A to G, same amino acid is
incorporated.
● Mutation Goes unnoticed – thus the name, “silent mutation”.

SILENT MUTATIONS

● In genetics, a missense mutation is a point mutation in which a

single nucleotide change results in a codon that codes for a
different amino acid.
● Single nucleotide change A to C-different amino acid
incorporated.
● Loss of functional capacity of protein.

MISSENCE MUTATIONS

● Single nucleotide change from C to T, stop codon is generated

(In mRNA represented by UAG), premature termination of
chain, may be incompatible with life.
● This type of mutation results in a shortened protein that may
 function improperly or not at all.

NON SENSE MUTATION

● Insertion or removal of a bases can alter the reading frame

with the resultant incorporation of different amino acids.
● Due to the triplet nature of gene expression by codons, the
insertion or the deletion can change the reading frame.
FRAME SHIFT MUTATION Resulting in a complete different translation from the original
one.

DNA EXTRACTION

DNA IS THE BLUEPRINT FOR LIFE.

everything living could contains DNA
● DNA isolation is one of the most basic and essential techniques in the study of DNA.
● The extraction of DNA from cells and its purification are of primary importance to the field of
biotechnology and forensics.
○ biotechnology in a sense that genetic manipulation of microorganism for the production of
antibiotics, hormones, and etc.
○ forensics tests are used in the detection of a crime.
● Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that
allows scientists to detect genetic disorders.
○ As discussed last week, through genetic counselling can foresee what characteristics or
traits can be passed down to the next generation.
● Produce DNA fingerprints of individuals
○ mentioned last week, can be used in crime scenes. So you can know who the suspect is
through the identification of the fingerprint
● Create genetically engineered organisms that can produce beneficial products such as insulin,
antibiotics, and hormones.

How can we extract DNA?

JUST FOLLOW THESE 3 EASY STEPS:
● Detergent
● Enzymes (meat tenderizer)
● Alcohol
OBJECTIVE:
● To extract DNA from cells.

MATERIALS:
● Blender ● Strainer
● Split peas (or any food you wish to extract the ● Meat tenderizer
DNA) ● Alcohol
● Salt ● Test tube
● Detergent ● Glass stirring rod
● Water ○ But you can improvise anything you
● Measuring cup and spoons can find in you home

PROCEDURE: (Disclaimer!! You may use other methods/sources for your final project)
1. The blender separated the pea cells from each other, so you now have a really thin peacell soup.
○ Assuming that we are using pea cells for our experiment.
2. Measure about 100 ml or ½ cup of split peas and place them in a blender.
3. Add a large pinch of salt (less than 1 ml or about ⅛ teaspoon) to the blender.
4. Add about twice as much cold water as the DNA source (about 200 ml or 1 cup) to the peas in the
blender.
5. Blend on high (lid on) for about 15 seconds.

AND NOW, THOSE 3 EASY STEPS:

1. Pour your thin pea-cell sop through a strainer into another container like a measuring cup or beaker.
2. Estimate how much pea soup you have and add about 1/6 of that amount of liquid detergent (about
30ml or 2 tablespoons). Swirl to mix (carefully)
3. Let the mixture sir for 5-10 minutes.

(continuation of procedure)
6. The detergent captures the proteins and lipids of the cell membrane.
7. Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
8. Add a pinch of enzymes to each test tube and stir gently.
● If you stir too hard, you’ll break up the DNA and you don’t want to do that. Its harder to see if
aggressive ra kayka.
● The DNA and nucleus of the cell is moulded, folded, and protected by proteins. The meat tenderizer
cuts the proteins away from the DNA.
(Use meat tenderizer for enzymes. If you can’t find tenderizer, try using pineapple juice or contact
lens cleaning solutions)
9. Tilt your test tube and slowly pour rubbing alcohol (70-95%
isopropyl or ethyl alcohol) into the tube down the side so that it
forms a layer on top of the pea mixture. Pour until you have
about the same amount of alcohol in the tube as pea mixture.
10. All of the grease and the protein that we broke up in the first two steps move to the bottom, watery
layer
11. DNA will rise into the alcohol layer from the pea layer. You can use a glass stirring rod or a wooden
stick to draw the DNA into the alcohol.
12. Slowly turning the stirring rod will spoon (wrap) the DNA around the rod so it can be removed from the
liquid.

WHAT DOES THE DETERGENT DO?

● Detergent cleans dishes by removing fats

○ It pulls apart fats or lipids and protein that make up the membrane surrounding the cell and
nucleus.
● It acts the same way in the DNA extraction protocol
● Once these membranes are broken apart, the DNA is released from the cell.
 WHAT DOES THE ALCOHOL DO?

● The DNA released from the cell nucleus is dissolved in the water/detergent/wheat germ solution and
cannot be seen.
● DNA precipitates (separates) out of solution in alcohol, where it can be seen.
○ Besides allowing us to see the DNA, the alcohol also separates the DNA from other cells
components which are left behind in the water solution.

RECAP:

Step 1: Blender insanity!

● The split peas cold water then blend it for 15 seconds. The blender separates the peas
cells from each other and you now have a really thin peacell soup.

Step 2: Soapy peas

● Pour your thin peascell soup through a strainer and add detergent. The detergent pulls
apart from the fats or the lipids and proteins that make up the membrane surrounding
the cell and nucleus.
● Only these membranes break apart, the DNa is released from the cell.

Step 3: Enzyme Power

● Add a pinch of enzyme and stir gently
● BE CAREFUL, if you stir too hard you’ll break up the DNA making you harder to see.
● For enzyme, you can use meat tenderizer, if none, try to use pineapple juice or contact
lens solution.

Step 4: Alcohol Separation

● You add alcohol, tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl
or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea
mixture.
● Alcohol is less dense than water, so if it floats on the top, look for clumps of white
strainy staff where the water and alcohol layer meets.