end of pathway
review
Test Thursday, December 10,
2015
Standard 1
Jimmy and Lukas
Objective 1
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500 B.C.: In China, the first antibiotic, moldy soybean curds, is put to use to treat boils.
A.D. 100: The first insecticide is produced in China from powdered chrysanthemums.
1761: English surgeon Edward Jenner pioneers vaccination, inoculating a child with a viral smallpox vaccine.
1870: The first experimental corn hybrid is produced in a laboratory.
1911: American pathologist Peyton Rous discovers the first cancer-causing virus.
1928: Scottish scientist Alexander Fleming discovers penicillin.
1933: Hybrid corn is commercialized.
1942: Penicillin is mass-produced in microbes for the first time.
1950s: The first synthetic antibiotic is created.
1958: DNA is made in a test tube for the first time.
1978: Recombinant human insulin is produced for the first time.
1979: Human growth hormone is synthesized for the first time.
1980: Smallpox is globally eradicated following 20-year mass vaccination effort.
1980: The Exxon oil company patents an oil-eating microorganism.
1982: The first recombinant DNA vaccine for livestock is developed.
1982: Human insulin produced in genetically modified bacteria, is approved by FDA.
1986: The first recombinant vaccine for humans, a vaccine for hepatitis B, is approved.
1988: The first pest-resistant corn, Bt corn, is produced.
1992: FDA approves bovine somatotropin (BST) for increased milk production in dairy cows.
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1994: The first breast cancer gene is discovered.
1994: The Americas are certified polio-free by the International Commission for the Certification of Polio Eradication.
1996: The first genetically engineered crop is commercialized.
1997: A sheep named Dolly in Scotland becomes the first animal cloned from an adult cell.
2003: China grants the world’s first regulatory approval of a gene therapy product.
2003: The Human Genome Project completes sequencing of the human genome.
2005: The Energy Policy Act is passed allowing for biofuel to be used.
2006: FDA approves the first HPV vaccine
2009: FDA approves the first genetically engineered animal.
2014: Magnet students amplify DNA.
Objective 2
Many question how ethical it is to extend human life
through artificial means. Many are against genetic
modifications of organisms. However, genetic
studies and modifications from studies such as the
Human Genome Project have allowed us to further
understand and treat the genome of multiple
organisms for certain ailments.
3. Know how to explain biotechnological concepts
effectively. Such as, “Polymerase Chain Reactions
allow us to amplify genes from a sample of DNA to
compare with others.” Or, “Gel Electrophoresis
allows us to create a gel DNA fingerprint to analyze
DNA.”
Objective 3
Career paths in biotechnological field:
Forensic analysis
Bioforensics
Bioinformatics
Biomaterials
etc.
Recall our guest speaker Chuck Folgogren of
Georgia BioEd Institute, who discussed data
analytics.
STANDARD 2
Brandon, Justin, Cameron, David
Personal Protective Equipment
• Safety goggles, gloves (and hot gloves), lab
coat, close-toed shoes, etc
• Protect the wearer from hazards in the lab
• Change depending on environment / materials
being used (for example, use hot/cold-resistant
gloves when dealing with extremetemperatures)
How to Safely Remove Gloves
The correct way to remove gloves:
Standard 2 : Objective 2
1. Autoclave- a machine that uses pressurized
steam to kill most microorganisms. The
standard conditions for an autoclave is usually
121゜C and 15-20 pounds/square inch. A
crockpot can be used as a subsitute for an
autoclave.
Standard 2: Objective 2
1. Disinfecting with Bleach- use 10% bleach to
disinfect countertops and used to clean
container tops in bleach baths. Bleach will kill
DNA.
2. Aseptic techniques- includes using gloves at all
time, a flame to circulate the air, and 70%
alcohol to spray on the countertop. Flaming
inoculation loops is also needed.
Standard 2: Objective 3
Appropriate Laboratory Behavior
• No horseplay in the laboratory
• Maintain aisles clear of objects
• No food or beverages in the laboratory
• “Read before you proceed”
Dangers of Contamination via Food & other
Objects
• Beverages may be confused with chemicals
• Food may have been contaminated by
chemicals
• Lotion, cosmetics, eye drops, and contact lenses
may pose potential health hazards while present
in the laboratory
Proper Disposal/Clean-up Procedures
• Report any accidents to your supervisor and
follow his/her instructions
• Some chemicals are to be put in the drain with
copious amounts of water
• Broken glass not to be touched
• Biohazardous material to be sterilized
Emergency Exits and Equipment
• In case of emergency follow your supervisor’s
instructions
• The emergency exit for this classroom is a door
found in the back of the room
• At the front of the room is an emergency
shower and eye wash
Emergency Exits and Equipment Continued
• Ask your instructor as to where emergency
equipment such as the fire extinguisher and
blankets may be found
Proper use of lab equipment
- Micropipettes:
- different sizes for measuring different
amounts of liquid
- a pipet least accurate when measuring at the
bottom of its range
- use fresh tip every time
Proper use of lab equipment
- Centrifuge:
- always balance centrifuge tube with another
tube of the same volume on the opposite side
- wait to open the chamber until the centrifuge
has come to a complete stop
Proper use of lab equipment
- Spectrophotometer- A sensitive instrument that
measures the amount of light at a specific
wavelength passing through a medium, usually
using cuvettes
- Give time to warm up before use
- Make sure cuvettes are inserted with clear side
facing the light
Personal Use of Lab Equipment
-pH meter
-A device that measures pH of a solution (acidity
or alkalinity).
- Do not contaminate, rinse after measuring each
reagent
Proper use of Lab Equipment
-Electrophoresis Apparatus
-The negative electrode (black) should be
nearest to the wells when placed in the chamber.
-Be sure to hook up the anode (-) and the
cathode (+) to their appropriate power supply.
-Pour buffer until wells are covered.
Micropipettes
- Micropipettes require disposable tips, so the tips
must be exchanged for a new one every time to
prevent contamination.
- Micropipettes have two push points on the top,
the first point of resistance is to obtain fluid and
dispose of fluid, the second point of resistance
is to blow out remaining fluid.
Proper use of lab equipment
-Thermal cycler
-Set the thermal cycler to the appropriate
program.
-Place PCR tubes into the block.
-Set the specific program for the reaction on the
thermal cycler’s interface.
Proper use of lab equipment
-Microscope
-Move the microscope slide around so that the
image is in the center of the field of view.
-Adjust the magnification to optimize image
quality
Proper use of lab equipment
-Autoclave
-Place autoclave tape over anything being
autoclaved.
-Refill water if meter is below proper level.
-If autoclave tape has black stripes, it has already
been autoclaved.
Proper use of lab equipment
-Balance
-Use proper weighing dishes when balancing a
solid
-Do not overstress the balance
Proper use of lab equipment
-Water Baths
-Be very careful when using a hot water bath to
avoid burning hands.
-Use a floating rack to place microcentrifuge
tubes in while in the hot bath.
STANDARD 3
Lena, Ashley B, Ethan
Objective 1.1: Follow Laboratory Protocols
● Following laboratory protocols is essential as a
mistake made in a lab could cause the lab to
fail. All mistakes are made in the lab notebooks.
Objective 1.1 (Continued), Objective 1.2
● One such mistake that caused a lab to fail was
in lab 7.5 (Biofuel Enzyme Assay). The
standards we produced were supposed to have
enzyme in them, but since the protocol never
called for it, enzyme was never added, leaving
us with clear standards.
Objective 2.1: Comply With Policies
• Companies must adhere to Industry Standards
• Industry standards are international
organizations that make sure products are
researched correctly and consistently. They also
certify that businesses processes fit an explicit
set of standards.
Objective 2.1 (Continued)
• These standards are called a quality system.
• Each company chooses the standards it will
meet. The Quality Assurance Department
within each company makes sure the standard
are meet.
Objective 2.1 (Continued)
• Records must be keep on the operations
happening in the company.
• Standard Operating Procedure(SOP) is followed
by everyone in the company.
• This is anything from a general business
procedure to a lab procedure.
Objective 2.2
• Good laboratory practice (GLP) is a quality
system used for non-clinical health health and
environmental studies.
• GLP is used to determine how to be safe in a
lab.
Objective 2.2 (Continued)
• Good manufacturing practice (GMP) Is a set of
principles for ensuring the quality and safety of
manufactured products used in health care.
• SOP, GLP, and GMP are enforced by the FDA
in the U.S. and European countries.
Objective 2.2 (Continued)
• Good documentation practices (GDP) are the
way a company records their data and any other
information.
• Each company tends to do it differently. The
correct way of recording for each company is
found in their SOP for recording data.
Objective 3.1
● All containers should be labeled immediately
during laboratory procedures. It is also a good
practice to pre-label containers, prior to adding
their contents.
● It is important to display health and safety
information on the labels of potentially dangerous
chemicals to avoid accidents and injuries.
Objective 3.2
● Labeling protocols for chemicals:
○ Name of solution
○ Concentration of solution
○ Date of preparation
○ Name of person who made solution
Objective 3.2 (Continued)
● Safety information for chemicals:
○ Health Hazards (coded blue)
■ 0 - Normal Material
■ 1- Slightly Hazardous
■ 2- Hazardous
■ 3- Extreme danger
■ 4- Deadly
Objective 3.2 (Continued)
○ Fire Hazards (coded red)
■ 0- Will not burn
■ 1- Above 200 F
■ 2- Below 200 F
■ 3- Below 100 F
■ 4- Below 73 F
Objective 3.2 (Continued)
○ Reactivity (Coded yellow)
■ 0- Stable
■ 1- Unstable if heated
■ 2- Violent chemical change
■ 3- Shock and heat may detonate
■ 4- May detonate
Objective 3.2 (Continued)
○ Specific Hazards (Coded white)
■ OXY: Oxidizer
■ ALK: Alkali
■ COR: Corrosive
■ ☢: Radiation hazard
Objective 3.3
● Material Safety Data Sheets are documents
presenting the potential dangers and hazards of a
chemical, as well as correct protocols for how to
handle the chemical.
● They Include:
○ Chemical details
○ Manufacturer contact/location information
○ Hazardous ingredients warnings
Objective 3.3 (Continued)
○ Physical/ Chemical characteristics
■ Boiling point, melting temperature, etc
○ Reactivity, Health, and Fire information
○ Control/disposal measures
○ Primary routes of entry
○ Emergency and first aid procedures
STANDARD 4
Chayton, Kaleigh
Standard 4: Objective 1
• 1) Molecular weight: the mass of one mole of a substance
Formula weight: the sum of the atomic weights of everything
that is in that particular formula (such as C6H12O6 being the total
weight of all the carbon, hydrogen, and oxygen atoms).
• 2)Ionic bonds form when two atoms have a large difference in
electronegativity (ex. Salt)
Covalent bonds form when two atoms have a very small
difference in electronegativity (complete sharing of electrons)
Hydrogen bonds is when hydrogen bonds with either fluorine,
oxygen, or nitrogen (H20)
• 3) Carbohydrates: any organic molecule including sugars, starch
and cellulose. They can be broken down to release energy.
(monosaccharides)
Lipids: any class of organic compound insoluble in water made
up of fatty acids. (store energy)
Proteins: nitrogenous organic compound consisting of large
molecules made up of long chains of amino acids. Essential to all
living organisms, i.e. structure.
Nucleic acids: complex organic substance present in living cells
(DNA/RNA). Whose molecules consist of many nucleotides
(GCATU)
• 4) Hydrophobic: tendency to not mix with water
Hydrophilic: having a tendency to mix with, dissolve in, or be
wetted by water
• 5) Acid base chemistry is adding acid to base so it won’t explode in
your face. Acids provide hydrogen ions (H+ ) while bases produce
hydroxide ions (OH-) in solutions.
• pH scale tell us how acidic or basic a solution is, the scale ranged
from 1-14 with 1 being acidic and 14 being basic, like. Water is
commonly at 7 aka neutral.
• Buffer’s function is the prevention of rapid change in pH when
acids or bases are added to a solution, and some properties include
solubility and pH.
Objective 2
• 1) We use the metric system to in biotechnology to measure things.
(cm, g, nm, µg, µl, k, °C, and j)
• 2) Molarity: moles/liters
• % volume per volume: (amount of liquid x or b/total amount of x+b)100
• % weight per volume: (weight/total amount of x+b)100
• 3) C1V1=C2V2. One concentration (C1) is the concentration of solution
one and the other concentration (C2) is the concentration of solution
two. One volume (V1) is the volume of solution one and the other
volume (V2) is the volume of the other solution. The number can be
interchangeable by which variable is missing. [ex. (2)(3)=(1)(x)]
• 4) We use indicators to indicate whether something is acidic or basic
such as phenolphthalein and bromothymol blue.
• 5) To label correctly you must put your initials, date, the concentration
of solution, and reagents. (If agar you must put what type, such as LB
agar).
• 6) When you prepare solutions make sure you have the correct
measurements and reagents required, make sure you add acid to base.
Also make sure the calculations are correct. If you add a powder or
‘solid’ to a liquid, add the solid about half of the solvent, then dissolve
and raise to total amount.
Objective 3
• 1) Dilution principals are the fact that many solutions in science are
more concentrated than desired. It is nessacary to dilution until the
concentration is desired. This requires working knowledge of
concentration factors and the calculations involved.
• To calculate a dilution factor it is the final volume/ aliquot volume
• 2) To make a serial dilution, one must make a series of identical
dilutions at known values (1mL of stock solution into 9mL of distilled
water, then 1mL of this solution into 9mL of distilled water, repeat
until final volume is reached).
STANDARD 5
Sydney, Lily, Jonathan, Symone
Structures of Organelles
• Nucleus: a membrane bound organelle
containing RNA and DNA. The major function
of the nucleus is the control center of the cell, it
regulates all processes in the cell.
• Nucleolus: a small body within the nucleus that
contains RNA and is composed of protein. The
major function of the nucleolus is to rewrite
ribosomal RNA and combine it with proteins.
• Endoplasmic Reticulum: a network of tubular
membranes within the cytoplasm (rough E.R.
has ribosomes). The major function of the
endoplasmic reticulum is to transport
synthesized proteins to the golgi apparatus.
• Golgi Apparatus: a complex of vesicles and
folded membranes within the cytoplasm. The
major function of the endoplasmic reticulum is
to transport synthesized proteins to the golgi
apparatus.
• Mitochondria: it contains a double membrane,
the inner layer being folded inward to form
layers (cristae) and are found in large numbers
in cells. The major function of the mitochondria
is create energy in the form of ATP.
• Ribosomes: a complex made of 50+ proteins,
with its own complement RNA. The major
function of ribosomes are to synthesize
proteins.
• Lysosomes: contain enzymes that degrade
cellular waste. The major function of lysosomes
are to digest excess or worn out organelles, food
particles, and engulfed bacteria and viruses.
• Chloroplast: disk shaped organelles containing
chlorophyll, only in plant cells. The major
function of the chloroplast is to absorb sunlight
and convert water and carbon dioxide to
produce glucose.
Structures of Organelles
Prokaryotic Cells
Cell Sizes:0.2 -2.0 µm in
diameter × 1-10 µm (0.001 –
0.01 mm): 2-8µm
Prokaryotic cells are generally
don’t have a membranebound nucleus."Pro-karyotic"
is Greek for "before nucleus".
Many prokaryotes are used in biotechnology and
medicine to create recombinant plasmids and mass produce
things like insulin or create pGLO.Most prokaryotes
transfer DNA by conjugation or binary fission. In
conjugation prokaryotes share DNA through contact. In
binary fission two prokaryotes break off parts of
themselves and their DNA is combined, however this in
not like meiosis in humans. Humans get half of both of
their parents genetic information. Prokaryotic cells contain
all of the parent cells DNA from both parents
Eukaryotic vs Prokaryotic
Eukaryotic Cells In Comparison to Prokaryotic
Cells
● cell size: eukaryotic cells range between 10 and 100 micrometers in
diameter, much bigger than prokaryotic cells which peak at 10 micrometers.
● cell membrane: composed of a phospholipid bilayer. They tend to be much
more selectively permeable than prokaryotic cells.
● genetic material: genetic material of eu. cells tends to be more so enclosed
and protected than prokaryotic cells, containing their genetic material in a
nucleus.
● The majority of euk. cells contain a nucleus, mitochondria, ribosomes, golgi
apparatus, lysosomes, and endoplasmic reticulum, varying much more
greatly in their organelles than pro. cells tend to. Euk. cells are much more
complex and diverse than pro. cells in general.
STANDARD 6
Ashley A, Andrea, Raiden
Standard 6: Objective 1
Agar as a media for growth:
★ solid: petri plates, slants, deep tubes
★ liquid: broth-keeps large quantities of
bacteria suspended & not for colony isolation
Bacteria incubate best at 37°. Agar media can contain
antibiotics such as penicillin to select resistant
bacteria. Disk diffusion tests can be used to test the
various effectiveness of antibiotics on bacteria.
S6:Objective 2: Inoculate Agar and broth media
-Different methods of inoculation
-Petri plates, Deep tubes, slants, solid and liquid
media are just a few different methods of
inoculation, and are all extremely common types
used for a variety of experiments.
Objective 2: Inoculate Agar and broth media
-Select appropriate media and methods of
inoculation
-Using wrong media and/or inoculation methods
can result in a failed experiment, wasted time, and
wasted resources.
Objective 2: Inoculate Agar and broth media
-Inoculate media using various techniques(i.e.,
steak and spread)
-These are techniques such as the streak and
spread, stabs for deep tubes, general aseptic
technique are all common techniques used
Objective 3: Identify common categories of
bacteria
1. BACTERIAL PROPERTIES:
● Domain of Prokaryotic Microorganisms
● various shapes: rods, spheres, and spirals
● inhabit most environments
● first lifeform on Earth
morphology
● spherical=cocci, rod=bacilli, spiral=spirilla
● single or multicellular
● smallest= 0.3 micrometers, largest= ½ millimeter
cell wall composition
● A cell wall is a layer located outside the cell
membrane
● peptidoglycan cell wall composed of disaccharides
and amino acids
● often a target for antibiotic treatment
metabolism
● autotrophic
GRAM STAINING
● distinguishes between components of cell walls
● gram positive vs. gram negative
POSITIVE: bacteria that give a positive result in the Gram stain
test.
NEGATIVE: bacteria that do not retain the crystal violet stain
STANDARD 7
Alaina, Amelia, Destyni, Bella
Standard 7
• Objective 1:
1. There are three components of nucleotides, deoxyribose (a sugar),
a phosphate, and one of the four bases (AGCT)
2. - RNA consists of a single polynucleotide strand, while DNA has two
Nitrogenous Bases:
RNA: Adenine, Guanine, Cytosine, and Uracil
DNA: Adenine, Guanine, Cytosine, and Thymine
- RNA are found in nucleus and cytoplasm of cell; DNA are mostly confined to chromosomes in nucleus a
- There are 3 types of RNA : mRNA, rRNA, tRNA; DNA have only one type
Objective 1:
3. Because DNA is
negatively charged it will
be pulled down toward
the positive end of the
gel, giving us our end
result with different
bands.
4. Restriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or RE’s). Restriction Digest is most commonly seen being used in
Restriction Fragment Length Polymorphisms (RFLP).
Objective 2:
1. - In DNA replication double stranded DNA is unwound and then separated into single template strands.
- Next short strings of nucleotides (primers) are bound to the template DNA so that DNA polymerase can b
- A DNA polymerase adds nucleotides to the 3’ end of the primer to extend the new complementary DNA s
- Key terms to remember: Lagging (Okazaki fragment), DNA ligase, leading strand, lagging strand, primas
Objective 2:
2. To perform PCR one of the things you need is template DNA which you get through the DNA replicatio
Primers are designed based on the sequences at the end of each target DNA
In both PCR and DNA replication the DNA is broken down.
3. PCR is defined as a Polymerase Chain Reaction and it is a simplified version of bacterial DNA replicati
There are 3 stages of PCR: Denaturation, Annealing, and Extension.
Gel electrophoresis is when DNA gets ran through an agarose gel to see the bands in DNA. From there we
4. Specialized equipment is needed to perform a PCR reaction such as a thermal cycler.
Components of a PCR reaction: Template DNA, nucleotides (dNTPs), PCR buffer, water, forward and reve
PCR is very sensitive and is prone to contamination, so strict sterilization protocols must be followed.
Reagents must be kept on ice until the reaction is ready to be placed in the thermal cycler.
Objective 3:
1. - Primary Structure: amino acid sequence in
the polypeptide chain
- Secondary Structure: Generated when
hydrogen bond between amino acids in close
proximity form two regular structures: alpha
helices and beta pleated sheets (alpha helices:
tight coils, beta pleated sheets: strands of 3-10
amino acids that lie side by side
- Tertiary Structure: the 3D shape of the
polypeptide chain when folded
- Quaternary Structure: reflects interactions
among multiple polypeptide chains, although not
all proteins have a quaternary structure
Protein
Structures
Function
Keratin
Skin, fur, nails
Covering and protection
Myosin
Thick filaments of
muscle fibers
Muscle contraction
Collagen
Skin, ligaments
tendons, and
extracellular matrix
Support
2. If a protein is
folded incorrectly it
will not be able to
function properly.
Examples in the
chart to the left.
Objective 3:
3. The classes of proteins are self explanatory and do just what their name implies.
-Support: cellular cytoskeleton
-Metabolism: enzymes and hormones
-Protection: antibodies
-Communication: signal transduction proteins that act as messengers between and
within cells
-Regulation: transcription factors
More examples below:
4. Answered
in picture
next to #1
Proteins in Biotechnology
Enzymes are used in
● industry to increase efficacy and cost
○ such as lipases, proteases, amylases, and cellulases
● life science research
○ such as PCR and ligases
● biochemical research
○ such as antibodies
Visualizing 3D protein structure
Using Protein Data Bank, Cn3D, and Chime
In 7.6, we used ClustalW to 3D view GFP
Picture of GFP protein from
Protein Data Bank
Separation techniques of proteins
SDS-PAGE coats proteins adjusting their charge to be relative to
their size (size exclusion chromatography
HIC chromatography separates proteins by using hydrophobic or
hydrophilic resin to separate based on charge
Ion exchange chromatography separates protein based on their net
charge. The resin can either be positively or negatively charged
Effects of Environment on the Function of
Enzymes
● Temperature and pH affect enzymes by denaturing the proteins
in extreme conditions
● Salt concentration affects the ionic structure of the protein
● Salt and pH can both be corrected using a buffer
How DNA becomes a trait
● DNA is replicated through mRNA and transcribed by tRNA.
This replication is continued by making a new chromosome and
eventually a chromatid. This DNA then becomes a trait
expressed through the cell.
Eukaryotic vs Prokaryotic Transcription
• Eukaryotic
• the exon and intron sequences are present in the initial
mRNA and is then processed to remove the exons and join
the introns in a process called RNA splicing. Post
translational modifications (additions of carbohydrate
molecules and phosphate groups) are much more common
in eukaryotes.
Eukaryotic vs Prokaryotic Transcription
• Prokaryotes
• In which regulated gene expression allows the cell to
respond to variations in the environment, such as changes in
the availability of nutrients.
Recombinant Protein
● Synthetic creation of proteins through the use of plasmids
● GFP is an example of a recombinant protein when expressed in
bacteria using a bacterial translation. The GFP is from the
pGLO plasmid and can be expressed easily in bacteria
Mutations
● Point mutations are mutations in just one or a few nucleotides
● Frame shift mutations are when the sequence is shifted over one
nucleic acid during tRNA transcription
● Nonsense mutations are premature stop codons in place of
amino acids
Consequences of mutations
● Mutations can cause chaos inside of a cell. If the mutation gets
fully transcribed, it can cause problems in formation of proteins
and health concerns.
● In extreme cases, mutations can end in a disrupted way of life,
or in the case of some genetic mutations and cancer it can lead
to death.
● Mutation problems vary for every person because everyone’s
genomic DNA is different
STANDARD 8
Maeva, Rachel, Taylor
The use of plasmids in Bacterial Transformation
Objective 1.1
Elements of a Functional Plasmid
Description
Origin of Replication
DNA sequence that allows the
start of replication within a
plasmid
Multiple Cloning Sites
Short segments of DNA that allow
for easy insertion of DNA due to
its several restriction sites
Promoter
Drives transcription of a target
gene by determining which cell
types the gene is expressed in and
amount of recombinant protein
obtained
Selection Gene
Gene of interest that is to be
introduced into a cell
The role of restriction enzymes
Objective 1.2-After PCR is used to create copies of a
gene from the genome of the organism, restriction
enzymes are used to cut the plasmid and open up the
circular piece of DNA allowing ligase to occur which
is where the gene of interest is linked. With ligation,
sugar phosphate bonds reform between base pairs and
the plasmid is formed into a circle again.
Competent Cells(8.1.3)
• Competent cells are bacterial cells that have
been altered and allow foreign DNA to be
passed into them easier.
• Bacteria become competent through the
addition of calcium chloride or through heat
shock.
Transformation(8.1.3)
• Once cells are competent, plasmid DNA is added to
cells and then cooled then heated rapidly. This allows
the cells to take on the DNA. The cells are then grown
on a culture that is incubated overnight after shaking
for 30 min. at 37 degrees celsius.
• Common transformation methods include
electroporation and transfection.
Selection(8.1.3)
• Transformed cells are selected by applying an
antibiotic to the agar. Bacteria that take up the
plasmid also express a resistance gene to the
antibiotic and will remain after the non
transformed bacteria are killed off.
Perform A Bacterial Transformation and Analyze Results(8.1.4)
• For the methodology behind transformation
and how to analyze results, please view the
previous three slides on the creation of
competent cells, transformation, and selection.
Analyze the protocol for isolating plasmid DNA
1. Pick up a colony of bacteria and inoculate it in your broth
media of choice containing your antibiotic (incubate overnight)
2. Pour culture into centrifuge tube and centrifuge it at desirable
speed and time
3. Discard the supernatant and wash the pellet. Centrifuge the
solution.
4. Discard the supernatant and resuspend the pellet in
resuspension solution. Mix to dissolve pellet.
5. Add Piper lysis solution into the tube and mix.
6. Pipet neutralization solution into the tube and invert.
Precipitate should form.
7. Centrifuge the tube and filter the supernatant into
microcentrifuge tube.
8. Spin column used
9. Fill the tube with wash solution and incubate
10. Put elution solution into tube
11. Microcentrifuge the solution
12. Store the miniprep plasmid DNA
How to quantify the amount of DNA purified
• Gel Quantification
• Real time PCR
• Spectrophotometric Quantification