CMB Laboratory

DNA Extraction and Gel

Electrophoresis

Prepared by RRL
(I) DNA Extraction

(II) Gel Electrophoresis

Laboratory
DNA or Deoxyribonucleic Acid
DNA,
the genetic material
Each chromosome contains one very long DNA
molecule with hundreds or thousands of
genes, each a section of the DNA of the
chromosome.
Transmitted from parents to offspring, genes
are the units of inheritance.
They encode the information necessary to
build all of the molecules synthesized within a
cell, which in turn establish that cell’s identity
and function.
What is DNA Extraction?
“Extracting DNA from the cell”

“Isolation of DNA by breaking the cell membrane with the help of chemicals,
enzyme or physical disruptions”

“Isolating nucleic acid from rest of the cell organelle”

We Extract DNA for
• The extraction of DNA is pivotal to biotechnology. It is the starting point
for numerous applications, ranging from fundamental research to routine
diagnostic and therapeutic decision-making.

• The ability to extract DNA is primary importance to studying the genetic

causes of disease and for the development of diagnostics and drugs.

• It is also essential for carrying out forensic science, sequencing genomes,

detecting bacteria and viruses in the environment and for determining
paternity.
The general
flowchart of the
DNA extraction
procedure
DNA Extraction
• CTAB or Cetyl trimethylammonium bromide serves as an important
surfactant in the DNA extraction buffer system to remove membrane lipids
and promote cell lysis. Separation is also successful when the tissue
contains high amounts of polysaccharides.

• SDS or sodium dodecyl sulfate is use as a chemical for proteins or urea for
nucleic acids. SDS is an anionic detergent that denatures secondary and
non–disulfide–linked tertiary structures, and additionally applies a negative
charge to each protein in proportion to its mass.

• Enzymes such as proteinase K, peptidase, protease disrupt proteins by

digesting it.
DNA Extraction
Once the cell wall or cell membrane lysed, there are no compartments inside
the cell. By adding Chloroform or phenol-chloroform and doing high-speed
centrifugation, DNA remains in the solution and the other cell debris settled
into the bottom of the tube.
Answer
• The aqueous phase is on top because it is less dense than the organic
phase (phenol:chloroform). The proteins and hydrophobic lipids will
partition into the lower organic phase while the nucleic acids (as well as
other contaminants such as salts, sugars, etc.) remain in the upper
aqueous phase.
DNA Extraction
• For DNA purification from detergents, proteins, salts and reagents used
during cell lysis step, the most commonly used procedures are: Ethanol
precipitation usually by ice-cold ethanol or isopropanol. Since DNA is
insoluble in these alcohols, it will aggregate together, giving a pellet upon
centrifugation. Q4
Answer
• Low temperatures protect the DNA by slowing down the activity of
enzymes that could break it apart. A cell's DNA is usually protected from
such enzymes (DNases) by the nuclear membrane which is disrupted by
adding detergent.
• Cold ethanol helps the DNA to precipitate more quickly.
• DNA is washed with 70% ethanol to remove some (or ideally all) of the
salt from the pellet. If water was used as the wash then DNA would
dissolve again and if 100% ethanol was used, the salt would not wash off
because sodium salts are poorly soluble in ethanol because precipitation in
100% ethanol cause removal of all.
DNA Extraction
TE or Tris-EDTA Buffer is a commonly used buffer solution in molecular
biology, especially in procedures involving DNA or RNA (the pH is usually
adjusted to 8.0 for RNA 8.0 and 7.5 for DNA). The respective DNA and RNA
nucleases are supposed to be less active at these pH values, but pH 8.0 can
safely be used for storage of both DNA and RNA.

DNA is pH sensitive and Tris Buffer maintains the pH of the solution.

EDTA or Ethylenediaminetetraacetic acid is a chelating agent and can

be used to block DNase activity. The chelator EDTA blocks the activity of
DNase by blocking the cofactor binding site. It works best in combination
with Tris.
CTAB Method
• Pre-warmed (65°C) 750 µL of CTAB solution and add approx. 100 mg of sample together with 50 µL of
20% SDS solution in 1.5 mL microcentrifuge tube.
• Each microcentrifuge tube containing the sample will mix thoroughly using vortex and incubate in digital
dry bath.
• The tube containing the solution will briefly cool down and 750 µL of chloroform alcohol will add to the
solution.
• The tube will mix using vortex.
• The tube containing the solution will subject to centrifugation for 10 min at 10,000 rpm.
• The aqueous phase will decant from the tube and transferred to a new microcentrifuge tube.
• Ice-cold isopropanol (0.5 ml, 1 volume) of 600 µL will add to the solution and will incubate for 30 min to
overnight at -20°C.
• The tube with solution will spin at 12,000 rpm for 10 min in centrifuge.
• The isopropanol alcohol will remove and the pellet will washed with 500 µL of 70% ethanol three times.
• The tube will spin again for 5 min at 7,500 rpm.
• The ethanol will remove by inverting the tube.
• The tube with the DNA pellet will air- dry and 50 µL of TE buffer will be added afterwards.
• Allow the DNA to dissolve by flicking the tube or heating using digital dry bath for 10 min at 65°C.
CTAB
Method
Equipment
 DNA and RNA Extraction

Q1 and Q2
DNA Extraction
DNA Extraction
DNA after DNA Extraction
Results of your Activity 1
Results of your Activity 1
Gel Electrophoresis
• It is a laboratory method used to separate mixtures of DNA, RNA, or
proteins according to molecular size.

• In gel, electrophoresis, the molecules to be separated are pushed by an

electrical field through a gel that contains small pores.

• The molecules travel through the pores in the gel at a speed that is
inversely related to their length.
Gel Electrophoresis
Gel Electrophoresis

• This means that a small DNA molecule will travel greater distance through
the gel than a larger DNA molecule.

• GE involves an electrical field such that one end of the has a positive
charge and the other ends has a negative charge.

• Proteins needed to be mixed with detergent called sodium dodecyl sulfate.

Gel Electrophoresis Machine
Steps in Gel Electrophoresis
Preparation of the Gel

Loading the DNA into the Gel

Gel Doc for formation of bands

Preparation of the Gel
• Using an erlenmeyer flask, solution of one percent agarose gel will prepare
by mixing agarose gel powder into Tris-acetate-EDTA(Ethylenediamine
tetraacetic acid) (TAE) Buffer solution (1x).

• The solution will mix and heat into microwave until the solution turns clear.
The liquid solution will cool down for about twenty minutes or until it is
tolerable to handle. (Approximately 2 to 4 ul of nucleic acid dye will be
added in the solution or Gel Red). The solution will then pour into the gel
tray. The comb will be place afterwards.

• The solidified gel will then remove in the gel tray and will place into the
Electrophoresis machine with TAE buffer solution (1x).
Gel Tray and Comb and Nucleic Acid Dye
Loading the DNA into the Gel
• Using micropipettor, one microliter (µL) of DNA from each samples will mix
into one microliter (µL) solution of five percent Bromephenol Blue and two
percent Gel Red.

• The mixed solution will be placed inside each hole of the solidified agarose
gel including the solution ladder which will also mix with one microliter
(µL) of five percent Bromephenol Blue and two percent Gel Red.

• The electrophoresis machine will then set to one hundred voltage and run
for thirty minutes before removing the agarose gel.
Gel Doc for formation of bands
• The agarose gel will then set into the Gel Doc to observe any formation of
bonds. The formation of bonds indicates the quantity and quality of the
DNA that can be subjected to amplification using PCR.
DNA Ladder
CMB Laboratory
DNA Amplification

Prepared by RRL
DNA Amplification
• In molecular biology, amplification is a process by which a nucleic acid
molecule is enzymatically copied to generate a progeny population with
the same sequence as the parental one.

• The production of multiple copies of a sequence of DNA.

• Repeated copying of a piece of DNA.

• The most widely used amplification method is Polymerase Chain

Reaction or PCR.

• Kary Mullis was awarded the Nobel Prize in Chemistry in 1993.

Thermal cyclers
• A thermal cycler is an instrument that automates temperature cycling and
incubation times for PCR. (PCR and qPCR)
Thermal Cyclers
Principle of PCR
• PCR is a technology used for quick and easy amplifying DNA sequences,
which is based on the principle of enzymatic replication of the nucleic
acids.

• Amplification is achieved by a series of three steps:

1. Denaturation, in which double-stranded DNA templates are heated
to separate the strands;
2. Annealing, in which short DNA molecules called primers bind to
flanking regions of the target DNA; and
3. Extension, in which DNA polymerase extends the 3’ end of each
primer along the template strands.

• These steps are repeated (“cycled”) 25-35 times to exponentially produce

exact copies of the target DNA
Three steps of PRC ̶ Denaturation, Annealing and Extension
Example of PCR Profile with 35 cycles
Step Temperature (°C) Time
Pre-denaturation 94 2 mins
Denaturation 94 30 secs
Annealing 51 to 55 30 secs
Extension 72 60 to 90 secs
Final Extension 72 5 mins
Hold 10 10 mins
Components of Master Mix
• DNA polymerase
• Primers (forward and reverse)
• DNTP mix
• Buffer
• Distilled/ Nano-pure/ Sterilized/ Nuclease-free water
• DNA

• Why do we need to do a Master Mix?

DNA polymerases
• DNA polymerases are critical components in PCR, since they synthesize the
new complementary strands from the single-stranded DNA templates.
• All DNA polymerases
possess 5’ → 3’
polymerase activity,
which is the
incorporation of
nucleotides to extend
primers at their 3’
ends in the 5’ to 3’
direction.
DNA polymerase
• The DNA polymerase used in
PCR are thermostable.

• One of the most thermostable

DNA polymerase is Taq DNA
polymerase, isolated from the
thermophilic bacteria species
Thermus aquaticus in 1976.
Primers
• Primers are short sequences of single stranded DNA that mark both ends
of the target sequence.
• Two primers are utilized, one for each of the complementary single strands
of DNA released during denaturation.
• The forward primer attaches to the start codon of the template DNA (the
anti-sense strand), while the reverse primer attaches to the stop codon of
the complementary strand of DNA (the sense strand).
• The 5’ ends of both primers bind to the 3’ end of each DNA strand.
Components of Master Mix
• Deoxyribonucleotide triphosphate or
DNTP Mix
• Each DNTP is made up of a
phosphate group, a deoxyribose
sugar and nitrogenous base.
• Contains sodium salts of dATP, dCTP,
dGTP and dTTP
• Buffer
• The buffer is usually between 8.0 to
9.5 and is often stabilized by Tris-
HCL.
Example of Master Mix
Component Concentration
Nuclease-free water 18.92 µL
10x of PCR Buffer 2.0 µL
10 mM of dNTP mix 0.4 µL
10 µM of forward primer 0.8 µL
10 µM of reverse primer 0.8 µL
Taq polymerase 0.08 µL
DNA 2 µL
Reaction Volume 25 µL
• The PCR products will be
checked using gel
electrophoresis for the
formation of bands.
The End ☺
• Please be ready of a Quiz.