end of pathway review

Test Thursday, December 10, 2015

Standard 1Jimmy and Lukas

Objective 1• 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.

• 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 analysisBioforensicsBioinformaticsBiomaterialsetc.

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 extreme-temperatures)

How to Safely Remove GlovesThe correct way to remove gloves:

Standard 2 : Objective 21. 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 21. 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

- 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

Proper use of lab equipment

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 4Chayton, 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 CellsCell 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 membrane-bound 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

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.

Standard 6: Objective 1

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 bacteria1. 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● heterotrophic

GRAM STAINING● distinguishes between components of cell walls

● gram positive vs. gram negativePOSITIVE: 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 twoNitrogenous Bases: RNA: Adenine, Guanine, Cytosine, and UracilDNA: Adenine, Guanine, Cytosine, and Thymine

- RNA are found in nucleus and cytoplasm of cell; DNA are mostly confined to chromosomes in nucleus and to mitochondria and chloroplasts (in small amounts)

- 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 bind to the double strands and begin to copy the template DNA- A DNA polymerase adds nucleotides to the 3’ end of the primer to extend the new complementary DNA strand - Key terms to remember: Lagging (Okazaki fragment), DNA ligase, leading strand, lagging strand, primase

Objective 2:

2. To perform PCR one of the things you need is template DNA which you get through the DNA replication process.

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 replication that copies a specific sequence of DNA.

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 are able to compare the DNA we run to the standard DNA.

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 reverse primers, and DNA polymerase.

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

CollagenSkin, ligaments tendons, and

extracellular matrixSupport

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 structureUsing Protein Data Bank, Cn3D, and ChimeIn 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 TransformationObjective 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 DNA1. 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 time3. 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 used9. Fill the tube with wash solution and incubate10. Put elution solution into tube11. Microcentrifuge the solution12. Store the miniprep plasmid DNA

How to quantify the amount of DNA purified•Gel Quantification•Real time PCR• Spectrophotometric Quantification