Bio Lab Final

What is a loading dye and what are its components

'Orange-G Loading Dye' is what we will use in this lab. 5X concentration so that the final concentration of the loading buffer becomes 1X when 10 μl of the loading buffer is added to 40 μl PCR product (1:4 ratio) making total volume of 50 μl COMPONENTS: 1. Dense substance to make DNA sample heavier when mixed - so that DNA sample sink on the bottom of the well in the gel 2. Tracking dye - for better visibility of DNA sample while loading on the gel - for approximation of the location of migrating DNA fragment

annealing

(bind) to primers in the target sequence The PCR reaction is cooled to a temperature that allows hydrogen bonds to form between the primers and the single-stranded template DNA. annealing temperature- temperature at which primers bind to the template - optimal annealing temperature is specific for each pair of primers (usually 50-60°C) and depends on the length and base composition of the primers. Annealing temperature is low enough for the primers to bind, but high enough to discourage reforming of the double-stranded template DNA. Because the primers are so much shorter than the template DNA, they will anneal much more quickly than the long template DNA strands at this temperature

transcription

(genetics) the organic process whereby the DNA sequence in a gene is copied into mRNA

ori

(origin of replication) product: region of specific sequence of DNA, where replication of plasmid DNA is initiated - Ori sequence does not code any protein products!!!!

Factors affecting mobility of DNA fragments through agarose gel

1. Agarose Concentration Higher concentrations of agarose facilitate separation of small DNA fragments, while low agarose concentrations allow resolution of larger DNA fragments. 2. Voltage As the voltage applied to a gel increased, DNA fragments migrate faster. However, as the voltage increase larger DNA fragments carrying larger net negative charge migrate proportionally faster than small fragments do. 3. Buffer Buffer solution must be used for both gel preparation and for running electrophoresis. The purpose of using buffer is: - to provide ions to support conductivity ( TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) - to establish pH ( so DNA molecules can carry a net charge)

transformation procedure

1. Suspend bacterial colonies in Transformation solution 2. Add pGLO plasmid DNA to a cell suspension 3. Incubate bacterial suspension with plasmids on ice for 10 min. 4. Heat-shock at 42°C for 50 sec and incubate on ice for 2 min. 5. Incubate bacteria in a nutrient broth for 15 min. at 37°C. 6. Spread bacterial cells on plates. 7. Culture bacteria on solid media for 24-36

Why perform each transformation step?

1. Suspension of bacterial cell in transformation solution: Transformation solution contains CaCI2. Positive charge of Ca++ions shields negative charge of DNA phosphates 2. Incubate on ice: Reduce the fluidity of cell membrane and maintains pore opening. 3. Heat-shock: Increases permeability of membranes. Foreign DNA molecules enter the cell by convection.(outside temp high; inside low) 4. Nutrient broth incubation: Need to allow beta-lactamase expression

requirements for a successful transformation

1. Viable cells to be transformed and appropriate support for growing, including a sterile condition to prevent contamination.We have a culture of actively growing E. coli cells and LB1 nutrient media. 2.Competence of the cells. Bacterial cells must be able to take up foreign DNA.To induce competency, we will apply a technique called 'heat-shock' to actively growing E. coli cells. In this process the bacterial cells are cooled in CaCl2 solution ('transformation solution') heated rapidly to create temperature gradient, which causes convectional influx of the solution containing pGLO. 3.Stably constructed plasmids containing a gene of interest.The plasmid we used (pGLO) contains a new gene (GLO), which encodes green fluorescent protein (GFP). 4.Selectable MarkersThe pGLO contains bla gene as a selectable marker of which product, -lactamase, digests ampicillin in the culture media to confer the bacteria resistance to the antibiotic. The cells that have not taken up pGLO will not survive on the LB nutrient media containing ampicillin. 5.Optionally, a reporter gene and associated regulator gene.This is a useful means to identify bacterial cells with the inserted gene turned on (i.e., with the expressed trait).On the culture media containing arabinose, the transformed E. coli cells turn GFP genes on and produce GFP protein, which fluoresces when illuminated with UV light. Therefore, the colonies can be easily identified by observing the culture plate under UV.We may collect the colonies that fluoresce under UV and culture them under an appropriate condition to produce a large amount of green fluorescent protein. Subsequently, GFP can be purified and characterized

Components of a PCR

1.Minute amount of double-stranded DNA template — containing the intact sequence of DNA to be amplified. 2.Individual dNTPs (deoxyribonucleoside triphosphates) — The dNTPs are the raw material of newly synthesized DNA and provide the power source for the reaction by the cleavage of phosphate groups. dNTPs are added as a mixture of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP); all of the dNTPs are added at the same concentration. 3.DNA polymerase — an enzyme that assembles the nucleotides into a new DNA chain. The first and most common DNA polymerase used in PCR is Taq DNA polymerase. 4.Magnesium ions (Mg2+) — added as MgCl2 and essential cofactor required by DNA polymerase to synthesize the DNA chain. 5.Oligonucleotide primers — pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies 6.PCR buffer — provides the optimum ionic environment and pH for optimal enzyme activity. Primers flank a target sequence giving the specificity to the PCR, but they are also necessary because DNA polymerase can only add nucleotides to double-stranded DNA.

A complete PCR amplification undergoes multiple cycles of PCR, usually ______ cycles.

25-40

transcription factor

A regulatory protein that binds to DNA and stimulates transcription of specific genes.

plasmid

A small, circular section of extra DNA that confers one or more traits to a bacterium and can be reproduced separately from the main bacterial genetic code.

master mix

A solution containing Taq DNA polymerase, primers, dNTPs, and a reaction buffer which is added to the DNA before temperature change

What is the name of the jellyfish used in lab?

Aequora victoria (IN ITALICS)

Factors affecting mobility of molecules through agarose gel

Agarose Concentration- You can resolve different sizes of molecules by using agarose at different concentrations. Higher concentrations of agarose facilitate separation of small molecules, while low agarose concentrations allow resolution of larger molecules Voltage Applied- As the voltage applied to a gel increased, charged molecules migrate faster. However, if larger molecules carry more net charges than smaller ones, they migrate proportionally faster than small molecules. Therefore, as higher voltage is applied, difference in distances migrated by small molecules and large molecules become smaller; thus, the resolution gets poorer Buffer-For agarose gel electrophoresis, a buffer solution must be used for both gel preparation and for running electrophoresis. The purpose of using buffer is: 1. to provide ions to support conductivity; i.e., ions in the buffer facilitate the flow of electrons (electric current). If deionized water is used instead of buffer, there will be no migration of charged molecules. Conversely, if you mistakenly use a buffer stock (concentrated buffer solution) without dilution, too much current will generate enough heat to melt the agarose gel. 2 .to establish pH so that molecules can carry a net negative charge, which is the driving force for migration of molecules in the agarose gel electrophoresis. The optimal acidity for electrophoresis for separation of molecules is the pH where the molecules take the highest electrical charge

What is agarose and what is agarose gel

Agarose- a mixture of carbohydrate polymers Agarose gel- a matrix (porous lattice) of the carbohydrate polymers filled with water In aqueous solutions, hydrogen bonding between polymers causes a gel to form at certain temperatures. Agarose has the melting temperature higher than gelling temperature. General purpose agarose forms gel around 35-40°C but melt at about 95°C; however, the gelling and melting temperatures can be affected by the concentration of the agarose If the temperature is too high before it becomes dispersed in the water, a shell of hydrated and/or gelled agarose can prevent rapid penetration of more water and can significantly slow dissolution melting of agarose should be started with water at room temperature.!!!!!!!!

Requirements for a good gel loading buffer

Also called a "gel loading dye" 1. Dense substance - to make DNA sample heavier when mixed. 2. Tracking dye - for visibility while loading sample and approximation of the location of migrating DNA fragments in the gel during electrophoresis 3. EDTA - inhibits action of DNA degrading enzyme (DNAse) - chelates Mg++ions, which is a cofactor required for the enzymatic action of DNase; thereby inhibiting action of DNase. 4. pH buffer at near neutral pH - to maintain negative charges on the phosphate groups of DNA for mobility during electrophoresis - Most common: Tris buffer @ pH 7.5

geneotype

An organism's genetic makeup, or allele combinations.

phenotype

An organism's physical appearance, or visible traits.

base pairs

Any of the pairs formed between complimentary bases in the two nucleotide chains of DNA, such as A-T and C-G (DNA); A-U and C-G (RNA)

enzyme-linked immunosorbent assay (ELISA)

Can detect the proteins that are produced specifically by GM crops. not useful for testing foods that have been highly processed, because the proteins have most likely been destroyed and different GM foods produce different proteins. PROS - quick and inexpensive CONS - crop specific, issue of protein stability

Components of electrophoresis apparatus

Common equipment includes dyes, trays, power supplies, electrodes, cables, gel mixtures, gel dryers, and chemicals such as denaturing agents, gel hardeners, and ampholytes

replication

Copying process by which a cell duplicates its DNA

Tracking dye vs. staining dye

DNA fragments in the gel are invisible without staining. •DNA is usually stained w/ a fluorescent dye (such as EtBror SYBR) and visible only under UV). •Without a tracking dye, we cannot tell where the DNA bands are migrating in the gel - no way to know if DNA fragments run off the gel. •Tracking dye in the loading buffer isnot for staining DNAmolecules. Tracking dye should not stain DNA since association with dye will change the molecular weight of DNA and affect the migration rate. - For actual visualization of DNA fragments, a fluorescent dye is needed. •A DNA staining dye SYBR - Safe is contained in both gel casting buffer and running buffer. •DNA fragments are stained while migrating through the gel in the buffer. - This staining method is compared to 'post-run staining', where DNA fragments are stained after running electrophoresis. •DNA fragments stained with SYBR - Safe are visualized

Master Mixes of PCR

DNA polymerase, dNTPs, primers, and reaction buffer containing magnesium ions are often combined into a master mix. A master mix is a bulk preparation of enzymes and buffers required in each of a series of reactions. Use of a master mix is highly recommended because it eliminates the need for transferring small volumes (some < 1μl) of each component to each reaction tube, which can introduce measuring errors. A master mix also ensures that the components in the reaction are present in exactly the same concentration in each reaction. To set up a PCR, a master mix containing the DNA polymerase, dNTPs, primers, and reaction buffer is added directly to template DNA in the PCR tube.

elongation

Extend the primers by reading the complimentary template and by adding matching nucleotides to the 3' end of the primer. The DNA polymerase binds to the region of double-stranded DNA created by the binding of the primer to the template DNA. The DNA polymerase reads the template strand in the 3' to 5' direction and adds nucleotides (A, T, G, or a C) one at a time at the 3' end of the primer to create a complementary copy of the original DNA template, extending in the 5' to 3' direction. The most common DNA polymerase used in PCR, Taq DNA polymerase, functions best at 72°C, so the extension step of PCR is usually conducted at 72°C.

Been around for centuries

Farmers have been genetically modifying crops for centuries and crop breeding to encourage specific traits, such as high yield, is still an important part of agriculture today. However, there is now the option to place genes for selected traits directly into crop plants. These genes do not have to originate from the same plant species—in fact, they do not have to come from plants at all.

Escherichia coli

For this lab.... You will learn how to introduce plasmid DNA containing a gene that codes for green fluorescent protein (GFP) into a special strain of the human symbiotic bacterium Escherichia coli. induce competence to E. coli bacteria enabling them to take up foreign DNA The E. coli cells that have taken up pGLO can produce -lactamase to digest ampicillin added in LB nutrient agar media and survive. As E. coli cells that have not acquired enough amount of functional pGLO cannot survive on the medium containing the antibiotic ampicillin, selection for cells that have taken up pGLO is accomplished by culturing them on the media plate containing the antibiotic. E. coli cells that contains no or non-functional GFP or araC gene will not glow under UV when cultured on the media containing arabinose, although they can survive on the media containing ampicillin.

GMO

Genetically modified organism - made when DNA is removed from one organism and placed within the DNA of what can be a very different organism.

denaturation

Heat the reaction to 94°C to separate (or denature) the double-stranded DNA molecule into two single strands. The high temperature causes the DNA double helix to separate by breaking hydrogen bonds between base pairs, resulting in single-stranded DNA.

Location of GMO's

In Europe, where GMOs are restricted in most countries in the region. However, GMOs are widely produced and sold in the United States. Currently in the US, foods that contain GMOs do not have to be labeled as such.

reporter gene

In addition to bla gene as a selectable marker, pGLO also contains a reporter gene, which tells you whether the gene of interest inserted in the plasmid is actually expressed and confers a new trait on the transformed cells. In this transformation experiment, the GFP gene, which itself is the new gene to be introduced in E. colicells, also serves as a reporter gene in conjunction with araC gene, which functions as an on/off switch for the GFP gene. (Refer to next section and Appendix D to see how the 'on/off switch' for the gene works.)In practice, it is rare to transform an organism with GFP gene alone. It is more often used as a reporter gene, which serves as an indication of whether a certain gene has been taken up by and expressed in the cell or organism. GFP gene is attached to another gene of interest when the characteristics the gene confers on organisms are not easily identified and measured. When GFP gene is used as a reporter, it is attached to the end of the sequence of the gene of interest and so as its expression to be regulated together with the gene of interest.

Introducing Plasmids into bacterial cells

In this lab, we are using an artificial transformation techniques. You will learn how to introduce plasmid DNA containing a gene that codes for green fluorescent protein (GFP) into a special strain of the human symbiotic bacterium Escherichia coli (E.coli; Figure 6-2). This strain of E. coli has been genetically modified to knock out the genes that encode the restriction enzymes, which otherwise might digest the plasmids taken up by the cell.

What is an intercalating dye? Names of the tracking dyes commonly used in agarose gel electrophoresis for separation of DNA fragments

Intercalating dyes alter mass and flexibility of linear DNA fragments, and thus their mobility. Examples are such as ethidium bromide and SYBR® dyes.

Bacillus thuringiensis (Bt)

KNOW HOW TO SPELL IT popular class of GM crops has a gene from the soil bacterium Transgenic crops that contain a gene from the bacterial species Bacillus thuringiensis that allows a natural insecticide to be produced in the crop. protein is a delta endotoxin (kills corn borers)

How a mixture of DNA molecules in different sizes are separated based on their sizes

Linear DNA fragments, which carries net negative charges can be separated on the basis of their sizes and visualized using agarose gel electrophoresis Linear DNA molecules must slip through the pores in the lattice in order to move toward the positive pole. Larger fragments will be slowed down more than smaller ones, since the smaller ones can fit through the holes faster. As a result, a mixture of large and small fragments of DNA that has been run through an agarose gel will be separated by size

polymerase chain reaction (PCR)

Look for a DNA sequence common to GM foods. DNA is more resistant than proteins to processing and can be extracted from even highly processed foods. It is these GMO DNA sequences that we will be testing for in this laboratory!!!!!!!! PROS - identifiable different GM crops, DNA stability CONS - expensive, time consuming

pGLO

Name for the plasmid that contains the jellyfish gene that codes for the GFP and gives the bacteria resistance to an antibiotic and the ampicillin resistant gene

transcriptional regulation of gene expression

Often, a researcher may want to control when transformed bacteria to produce the protein of interest. Proteins called transcription factors are frequently used by cells to switch transcription "on" or "off" depending on environmental conditions. The transcription, or production of mRNA, of the GLO gene is controlled by using a promoter that is only active in the presence of the sugar arabinose. The AraC protein, encoded by the araCgene on the pGLO plasmid, is the transcription factor necessary for this control. This protein is bound at the pGLO promoter site, but without arabinose is in the incorrect conformation, or shape, to recruit RNA polymerase and initiate transcription In the presence of arabinose, the sugar binds to the AraC protein and changes its conformation so that, in combination with RNA polymerase, transcription is initiated and an mRNA transcript is produced. In bacteria, transcription and translation, or protein synthesis, occurs simultaneously, and the GFP protein is produced. Under the regulatory mechanism described above, the successfully transformed cells will appear white on plates not containing arabinose; but, will fluoresce green under UV when arabinose is included in the nutrient agar medium. However, E. coli cells that contains no or non-functional GFP or araC gene will not glow under UV when cultured on the media containing arabinose, although they can survive on the media containing ampicillin.

Facts on lb broth

Originally formulated by Bertani to study bacteriophage, and LB meant Lysogeny(cell-bursting) Broth • Later Luria modified the formula and now LB broth refer to Luria-Bertani broth • LB broth formula has been further modified by others .• LB broth we use has been formulated for bacterial growth and gene expression, containing: Carbohydrates Amino acids Nucleotides Salts Vitamins •LB agar media is solidified by adding 1% agar in LB broth.

What is PCR?

PCR is a simplified version of bacterial DNA replication that copies a specific sequence of DNA (the target sequence) so that it is amplified. The target sequence is replicated again and again to make millions of billions of copies. Copies produced by PCR are called PCR products or amplicons.

translation

Process by which mRNA is decoded and a protein is produced decoding of a mRNA message into a polypeptide chain

arabinose

SUGAR that enables protein expression NOT the protein!!!! Just the sugar

How to track DNA fragments migrating on an agarose gel

Since 'stained' DNA molecules are only visible under UV after completion of electrophoresis run, it is also necessary for us to use 'tracking dyes' to approximate the location of the migrating DNA molecules on the gel during electrophoresis. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appea

Final step of a PCR cycle is for a DNA polymerase to extend the primers by reading the complimentary template and by adding matching nucleotides to the 3' end of the primer.

The DNA polymerase binds to the region of double-stranded DNA created by the binding of the primer to the template DNA. The DNA polymerase reads the template strand in the 3' to 5' direction and adds nucleotides (A, T, G, or a C) one at a time at the 3' end of the primer to create a complementary copy of the original DNA template, extending in the 5' to 3' direction. The most common DNA polymerase used in PCR, Taq DNA polymerase, functions best at 72°C, so the extension step of PCR is usually conducted at 72°C. DNA polymerase adds These three steps of PCR—denaturation, annealing, and extension—comprise one cycle of PCR. A complete PCR amplification undergoes multiple cycles of PCR, usually 25-40 cycles. The entire 40 cycle reaction is carried out in a 0.2 ml thin-walled PCR microcentrifuge tube that has been placed in a thermal cycler or PCR machine. This is a machine that contains an aluminum block that can be rapidly heated and cooled. The rapid heating and cooling of this thermal block is known as thermal cycling

template strand

The DNA strand that provides the pattern, or template, for ordering, by complementary base pairing, the sequence of nucleotides in an RNA transcript. Two new template strands are created from the original double-stranded template during each complete cycle of PCR. This causes exponential growth of the number of target DNA molecules, i.e., the number of target DNA molecules doubles at each cycle (2^n, where n is the number of cycles); this is why it is called a chain reaction. Therefore, after 40 cycles there will be 2^40, or over 1,100,000,000,000 times more copies than at the beginning. Once the target DNA sequences of interest have been sufficiently amplified, they can be visualized using gel electrophoresis. This allows researchers to determine the presence or absence of the PCR products of interest.

The next step of the PCR cycle is to allow the primers to locate and anneal (bind) to the target sequence

The PCR reaction is cooled to a temperature that allows hydrogen bonds to form between the primers and the single-stranded template DNA. The temperature at which primers bind to the template is called the annealing temperature. The optimal annealing temperature is specific for each pair of primers (usually 50-60°C) and depends on the length and base composition of the primers. Annealing temperature is low enough for the primers to bind, but high enough to discourage reforming of the double-stranded template DNA. Because the primers are so much shorter than the template DNA, they will anneal much more quickly than the long template DNA strands at this temperature

Relationship between size of DNA fragment and migration distance on an agarose gel

The migration rate of a DNA sample is affected by many factors including..... - the electrophoresis apparatus used - width of gel well - volume and concentration of DNA sample loaded - voltage gradient and buffer strength, etc - the migration distance may different one run from another Therefore, at least one size marker must be loaded in a lane on the gel to estimate the size of the sample DNA fragment. When a wide gel is used, two lanes on the gel are usually loaded with size markers since migration rates of DNA fragments near one side may differ from ones on the other side

Many people object to the use of GM crop plants.

They argue that there is a potential to create super-weeds through cross-pollination with herbicide-resistant crops or that super-bugs will evolve that are no longer resistant to the toxins in pest-resistant crops. Many are concerned with potential allergic reactions to the novel proteins or antibiotic resistance arising from the selectable markers used to develop the crops or other unforeseen effects on public health. Proponents of GM foods argue these crops are actually better for the environment. Fewer toxic chemicals are put into the environment and thus fewer toxic chemicals can harm the environment and human health. In addition, these crops can preserve arable land by reducing stresses on the land, improve the nutritional value of food in developing countries, and allow crops to be grown on previously unfarmable land.

Difference between tracking and intercalating dyes.

Tracking dyes are different from DNA staining dyes in that tracking dyes do not associate with DNA molecules while DNA staining dyes bind DNA molecules with high affinity.

selectable marker

Transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells. Therefore it is necessary to select for the bacterial cells that have acquired the foreign DNA. A selectable markeris a gene incorporated into a plasmid that confers a trait suitable for selection of the transformed cells. One of the selectable markers most commonly used in bacterial transformation is the antibiotic resistance gene. The transforming plasmid contains a gene that confers bacteria resistance to an antibiotic that the bacteria are otherwise sensitive to; thus, those bacterial cells without the plasmid containing the gene may be killed or have their growth arrested. To select transformed cells, the mixture of cells, some of which have taken up the transforming plasmids while other not, is cultured on media containing the antibiotic so that only transformed cells are able to grow. The pGLO contains a gene (bla), which encode the enzyme -lactamase, which digests ampicillin in the culture media to confer the bacteria resistance to the antibiotic. The E. coli cells that have taken up pGLO can produce -lactamase to digest ampicillin added in LB nutrient agar media and survive. As E. coli cells that have not acquired enough amount of functional pGLO cannot survive on the medium containing the antibiotic ampicillin, selection for cells that have taken up pGLO is accomplished by culturing them on the media plate containing the antibiotic. Only cells that acquired pGLO will gorw to form colonies on the medium containing the antibiotic ampicillin

Bt crops

Transgenic crops that contain a gene from the bacterial species Bacillus thuringiensis that allows a natural insecticide to be produced in the crop.

induced competence

We will transitionally induce competence to E. coli bacteria enabling them to take up foreign DNA. When bacterial cells are made competent by an artificial treatments, they are called induced or artificial competent cells, compared to the naturally competent cells. To induce competence to E. coli cells, we will use a technique called 'heat-shock', in which actively growing bacterial cells are suspended in CaCl2 solution (transformation solution) and incubated at cold temperature (0°C) followed by warm temperature (42°C). The actively growing bacterial cells have pores on their membranes, which are large enough for small plasmids to pass through. However, as phosphate groups on both DNA molecules and phospholipid of the cell membrane are negatively charged, they repel each other. The Ca2+ ions in the solution shield negative charges on DNA molecules and phospholipids of cell membrane, neutralizing both. When the cell membrane is rigidified by the cold treatment, the lipid molecules in the membrane become thermodynamically stabilized making them easier for calcium ions to shield the negative charges. A rapid treatment with warm temperature ('heat-shock') quickly raises the outside temperature of the cell creating a temperature gradient so that the plasmids in the solution are carried into the cell by convectional current.

competency/competent

When bacterial cells are in a state of being capable of taking up the foreign plasmid, they are said to be competent, and those bacterial cells are called competent cells. The capability of bacterial cells to take up the foreign plasmid is referred to as competence. When a competent bacterium takes up a foreign plasmid that is beneficial to the bacterium, this natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.

competent cells

When bacterial cells are in a state of being capable of taking up the foreign plasmid, they are said to be competent, and those bacterial cells are called competent cells. The capability of bacterial cells to take up the foreign plasmid is referred to as competence. When a competent bacterium takes up a foreign plasmid that is beneficial to the bacterium, this natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.

Relationship between size of molecules and migration distance on an agarose gel

When electric current is applied through the gel and an electrical potential (voltage difference between plus and minus poles) is established, negatively charged molecules migrate toward the positive (red wire); positively charged ones toward the negative (black wire). Under the electrical potential, charged molecules must pass through the pores in the lattice and move toward the electrode. Larger molecules will be slowed down more than smaller molecules because the smaller molecules can pass through the holes faster. As a result, a mixture of large and small molecules that has been run through an agarose gel will be separated by size.

reporter genes

a gene whose phenotype is easily observed, used as genetic marker to make sure that you correctly inserted a gene

Taq DNA polymerase

a heat resistant enzyme found in the bacillus Thermus aquaticus, which lives in hot springs, that can endure the high temperatures (94°C) of the polymerase chain reaction.

Using a sterile pellet pestle attached to the motor, GMO will be grinder to resemble

a homogenate consistency of medium-thick soy milk

antibiotic resistance gene

a selectable marker that is most commonly used in bacterial transformation plasmid contains a gene that confers bacteria resistance to an antibiotic that the bacteria are sensitive to, those bacterial cells without the plasmid containing the gene may be kills or have their growth arrested.

How to estimate sizes of DNA fragments separated on an agarose gel

acking dye for visibility while loading sampl during electrophoresis

heat shock

actively growing bacterial cells are suspended in CaCl2 solution and incubated at 0 degree C temperature followed by 42 degree C temperature

reverse primers

anneal at the end of the targeted region

forward primers

anneals the beginning of the targeted region of DBA

ampicillin

antibiotic when cultured on the media containing arabinose, although they can survive on the media containing ampicillin

selectable markers

antibiotic resistance genes and other vector genes that make it possible to pick out cells harboring a particular DNA molecule used to ensure the recombinant DNA is taken up and retained by the host organism

ara vs araC

ara is arabinose araC is the protein

gene product trait

bt gene crystal kills corn borers

genetic transformation

change in a genetic trait of an organism caused by the insertion of a foreign gene into the organism's genome

Primer dimers

consists of primer molecules that have attached to each other because of strings of complementary bases in the primers.

Bt crops produce a protein called ____ that is lethal to European corn borers, a common pest on corn plants

delta-endotoxin When the corn borers feed on the genetically modified plant, they die. Other GMOs include those that are herbicide-resistant, delayed for fruit ripening, resistant to fungi or drought, have increased crop yield, or bear improved fruits.

Three Steps of PCR

denaturation, annealing, extension (or elongation)

Kary Mullis at Cetus Corporation

developed the technique that has since revolutionized molecular biology research. This technique, called the polymerase chain reaction (PCR), transformed molecular biology into a multidisciplinary research field within 5 years of its invention. Before PCR, the molecular biology techniques used to study DNA required such a high level of expertise that relatively few scientists could use them. PCR is now used as a medical diagnostic tool to detect specific mutations that may cause genetic disease. It is also used in criminal investigations and courts of law to identify suspects. The molecular biology techniques for therapeutic, forensic, pharmaceutical, agricultural, or medical diagnostic purposes was neither practical nor cost-effective. The development of PCR technology changed these aspects of molecular biology from a difficult science to one of the most accessible and widely used tools in biotechnology.

ELISA

enzyme-linked immunosorbent assay detect the proteins that are produced specifically by GM crops. NOT useful for testing foods that have been highly processed, because the proteins have most likely been destroyed and different GM foods produce different protein.

Estimation of molecular size of DNA

estimated by comparing distance it travels also called: size marker molecular marker molecular weight ruler DNA ladder

First and second lab sessions

first lab session you will extract genomic DNA from food samples second lab session you will electrophorese the amplified samples to visualize the DNA for analysis

thermostable

not easily altered, decomposed or destroyed by heat

LB broth

nutrient broth purpose: need to allow for beta-lactamase expression

Construction of pGLO

pGLO contains several genes that enable replication of the plasmid DNA and expression of the fluorescent trait (phenotype) in bacteria following transformation GFP — The jellyfish gene that codes for the production of Green Fluorescent Protein. •bla — A gene that encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin, and thus confers resistance to the antibiotic ampicillin. Bacteria containing the bla gene can be selected by placing ampicillin in the growth medium. Without the bla gene, bacteria are killed by the antibiotic ampicillin. •ori — The origin of pGLO plasmid DNA replication. This is a sequence of DNA at which replication of plasmid is initiated. •araC — A gene that encodes the araC protein that binds to a specific sequence of DNA (called a promoter). When arabinose binds to the araC protein, an arabinose-araC complex is formed.Only the arabinose-araC complex can bind to the promoter and the production of GFP is switched on. Thus, the gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells' nutrient medium.

oligonucleotide primers

pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies

PCR

polymerase chain reaction look for a DNA sequence common to GM foods. DNA is more resistant than proteins to processing and can be extracted from even highly processed foods.

transformation

process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria

objective of PCR:

produce a large amount of DNA in a test tube (in vitro), starting from only a trace amount Because PCR identifies a specific sequence of DNA and makes millions of copies of (or amplifies) that sequence, it is one of the most useful tools of molecular biology. Scientists use PCR to obtain the large amounts of a specific sequence of DNA that are necessary for such techniques as gene cloning, where DNA is physically moved from one genome to another.

bla

product: A gene that encodes the enzyme beta-lactamase. trait: breaks down the antibiotic ampicillin. - Bacteria containing the bla gene can be selected by placing ampicillin in the growth medium. - Without the black gene, bacteria are killed by the antibiotic ampicillin.

araC

product: arabinose C protein trait: regulates expression of GFP genes - also binds to promoter sequence of DNA

GFP

product: jellyfish gene that codes for the production of Green Fluorescent Protein trait: fluorescence under UV

delta endotoxin

protein that is lethal to corn borers, a common pest to corn plants. Farmers who plant Bt crops do not have to apply pesticide because the plants produce the toxic protein inside their cells.

gene

sequence of DNA that codes for a protein and thus determines a trait

Oligonucleotides

short nucleotide sequences that are used in PCR and as complementary genetic probes to identify specific gene sequences

DNA must be _____ before it can be replicated, so that each primer can anneal to the separated template strands

single-stranded So, the first step of PCR is heat the reaction to 94°C to separate (or denature) the double-stranded DNA molecule into two single strands. The high temperature causes the DNA double helix to separate by breaking hydrogen bonds between base pairs, resulting in single-stranded DNA.

How to visualize (stain) DNA fragments on an agarose gel

stained with a fluorescent dye (Ethidium bromide is a fluorescent dye most commonly used for staining nucleic acids -both DNA and RNA) ------> !!!!!!!!! Due to its potential hazard, we use a dye called SYBR® Safe1 in this lab exercise. SYBR® Safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can be used with either blue-light or UV excitation. Another advantage of SYBR® Safe stain is that it can be boiled with agarose, while ethidium bromide must be added after the molten agarose has cooled down below 60°C due to its toxic fume generated at higher temperature

Components of PCR

strength of PCR lies in its ability to specifically target a section of DNA within a much larger quantity of DNA PRIMERS- The sequence is targeted with two pieces of short, single strands of DNA designed to match and bind (anneal) each end of the target sequence. (1) first primer, called the forward primer, anneals the beginning of the targeted region of DBA (2) second primer, called the reverse primer, is designed to anneal at the end of the targeted region The two primers are designed and synthesized in the laboratory with a specific sequence of nucleotides to provide the specificity of PCR selecting the region to be amplified. Primers also serve as starting points for DNA synthesis by Taq DNA polymerase, which can bind itself and add nucleotides to the double-stranded DNA only. The DNA polymerase used in PCR must be stable at high temperature (themostable) because the PCR reaction is heated to 94°C to separate the DNA strands, which would destroy the biological activity of most enzymes. The most commonly used thermostable DNA polymerase is Taq DNA polymerase. This was isolated from a thermophilic bacterium, Thermus aquaticus, which lives in high- temperature steam vents such as those in Yellowstone National Park. The enzymes within these bacteria have evolved to withstand high temperatures (94°C) and can be used in the PCR.

CaCl2

transformation solution known as calcium chloride Significance: To induce competence to E. coli cells, we will use a technique called 'heat-shock', in which actively growing bacterial cells are suspended in CaCl2 solution (transformation solution) and incubated at cold temperature (0°C) followed by warm temperature (42°C).

induced or artificial competent cells

when calls are made competent by artificial treatment, such as a heat shock in bacterial transformation