Genetics Lab Exam PDF

**Genetics Lab Exam** 1. **Functional groups** - Know the chemical formula of the following functional groups - Amine- -NH2 - Carboxylic acid- -COOH - Amide- -CONH2 - Hydroxyl- -OH - Aldehyde- -CHO - Ketone- -CO- - Ether- -O- 2. **Macromolecule...

**Genetics Lab Exam** 1. **Functional groups** - Know the chemical formula of the following functional groups - Amine- -NH2 - Carboxylic acid- -COOH - Amide- -CONH2 - Hydroxyl- -OH - Aldehyde- -CHO - Ketone- -CO- - Ether- -O- 2. **Macromolecules: Know the monomeric unit, name of the bonds joining the monomeric units and identify the bonds when a structure is provided** - Carbohydrates - Types of carbohydrates: Know examples of each type of carbohydrate. Specific structures do not have to be memorized but students should be able to identify mono-, di-, oligo-, or polysaccharides if a structure is shown Monosaccharides-glucose; 6 carbon monosaccharide C6H12O6. Disaccharides-sucrose; 2 monosaccharides, oligosaccharides- 3-10 monosaccharides; raffinose, polysaccharide many monosaccharides, starch - Polymerization of carbohydrates Groups involved in the polymerization process and name of the bond Monosaccharides, connected through glycosidic bonds - Types of glycosidic bonds and how to name the glycosidic bond- Alpha glycosidic bonds- anomeric carbon below the O in glycosidic bond. Beta glycosidic bond- anomeric carbon above the O in glycosidic bond. - Lipids - Fatty acids: Difference between saturated and unsaturated fatty acids Saturated fatty acids do not have double binds, but unsaturated fatty acids do have double bonds. Cis unsaturated fats have the hydrogen of the double bond on the same side, but trans unsaturated fats have the hydrogen of the double bond on the opposite sides. - Types of lipids Glycerides- neutral and phosphoglycerides, steroids, wax - Bond that covalently attaches fatty acids to glycerol Ester bonds - Identify structure of mono-, di-, triglycerides, steroids, waxes, and phospholipids Steroids- contains sterol ring (4 carbon rings), wax- fatty acid to long chain alcohols - Phospholipids: Identify different parts what makes hydrophobic tail and hydrophilic head. Two fatty acids and one phosphate group attached to glycerol, hydrophobic tail = phosphate, hydrophilic head= fatty acid part. - Proteins - Monomeric unit of proteins Amino acids - Amino acids: How many are there, 3 letter name, 1 letter name, general structure 20 amino acids available - Polymerization of amino acids Groups involved in the polymerization process and name of the bond. Be able to identify the bond when an oligopeptide is provided -Two amino acids joined together through an ester bond, has a -NH2 and -COOH group - Type of reaction that helps in joining of 2 amino acids (dehydration)- Condensation reaction or dehydration synthesis reaction - **Different between oligo- and polypeptide- Oligo- 2-20 amino acids, poly- more than 20, can fold to form 3D structures.** - Be able to identify N- and C-terminus- N-terminus: beginning of the chain, marked by a free amino group (-NH2). C-terminus: end of the chain, marked by a free carboxyl group (-COOH). - Nucleic acids - Monomeric unit of DNA and RNA: parts of a nucleotide and where phosphate group and nitrogenous bases are attached to ribose or deoxyribose sugar.- Nucleotides made of phosphate, sugar( deoxyribose in DNA or ribose in RNA), nitrogenous bases. The nitrogenous base is attached to the 1' carbon of the sugar molecule, while the phosphate group is attached to the 5' carbon of the sugar. - Structures of purines, pyrimidines, ribose, deoxyribose, phosphate groups, nucleoside, and nucleotide: Students should be able to draw the structures as well as identify them if a structure is provided.- Nucleoside has no phosphate group, nucleotide does have a phosphate group. Purines have a double ring molecule (A & G). Pyrimidines have a single ring molecule (U in RNA,C, T in DNA). - Name of bond that joins nucleotides together to form oligonucleotides or polynucleotides- Phosphodiester bond -  Be able to draw 2 nucleotides joined together - Identify the 5'end and 3' end of oligo- or polynucleotide strand (chain)- 5' end: the end of the strand where the phosphate group is free, starting point of the strand in conventional notation. 3' end: the end of the strand where the hydroxyl group (-OH) on the 3' carbon of the sugar is free; this is the direction in which nucleotides are added during replication or transcription. - Types of nucleic acids- Ribonucleic acid, deoxyribose nucleic acid - Structure of DNA - - - - Structure of RNA - - - Differences between DNA and RNA- Feature DNA ------------------- ---------------------------- ------------------------------ Sugar Deoxyribose Ribose Nitrogenous bases A,T,G,C A,U,G,C Structure Double-stranded helix Single-stranded helix Stability More stable (lacks 2'-OH) Less stable (2' -OH present) Function Stores genetic information Various roles:mRNA,tRNA - Be able to draw a dinucleotide** ** 3. **Detecting DNA and RNA** - What is gel electrophoresis?- a lab technique used to separate and analyze molecules such as DNA, RNA, or protein based on their size and charge by applying an electric field through a gel matrix. - What type of matrix can be used for electrophoresis?- Agarose gel: commonly used for DNA and RNA. Polyacrylamide gel: used for proteins or smaller DNA/RNA fragments. - The two functions of the loading dye during gel electrophoresis.- Weight: provides density to the sample, ensuring it sinks into the wells. Visualization: contains tracking dyes to monitor the progress of the electrophoresis. - What is the purpose of 1X TBE in agarose gel electrophoresis?- Buffering maintains a stable pH during electrophoresis. Ionic conductivity facilitates the flow of electricity through the gel. - What charge does nucleic acids have? Why?- Nucleic acids are negatively charged due to the phosphate's groups in their backbone. - How would you know that electricity is running through the gel?- Visible bubbles at the electrodes indicate thru flow of electricity. Migration of tracking dye also confirms current flow. - By looking at the gel, how will you know whether you have intact RNA or not?- Intact RNA will show distinct, sharp bands. Degraded RNA will appear as a smeared pattern or lack defined bands. - What are the different bands observed in RNA?- Eukaryotic RNA is 28S and 18S rRNA bands. Small RNAs may also be visible depending on the gel resolution. - Name a dye used to stain DNA and view using a microscope- Ethidium bromide (EtBr) fluoresces under UV light. Alternative dyes: SYBR Green or GelRed. - Know how to identify a plant cell and animal cell- Plant: cell wall, chloroplasts, large central vacuole, regular rectangular shape. Animal: no cell wall, lack chloroplasts, small or absent vacuoles, irregular or round shape. 4. **Volumes** - Be able to identify Erlenmeyer (conical) flasks, serological pipettes, measuring cylinders, volumetric (standard) flasks, microcentrifuge tubes, beakers, and micropipettes. Know which one is used for measuring and which is used for storing or holding liquids.  - Know the units of mass, volume, and density.- Mass: grams, milligrams, kilograms. Volume: liters, milliliters, microliters. Density: mass per unit volume (g/mL, or kg/L) - Know how to appropriately use different sized micropipettes with the correct tip size.  - Know the difference between P10, P200, and P1000 micropipettes.- P10: measures 0.5-10 microliters. P200: measures 20-200 microliters. P1000: measures 100-1000 microliters. - Know when to use each micropipette depending on the given volume. - Know how to convert between different units of measurement. An example is milliliters to microliters and vice versa. - Know how to calculate percent error, averages, and standard deviations.- Percent error- (measured value- true value)/ true value x 100. Average: sum of all values/ number of values. Standard deviation: σ=[\$\\sqrt{}\$]{.math.inline}∑(xi​−xˉ)2​​/n. xi is each data point, x\` is the mean, and n is the number of data points. - Know how to use excel for data transfer and interpretation. Be able to use Excel to calculate average and standard deviations using Excel. 5. **Bacterial Transformation** - What is horizontal and vertical transfer of genetic information- Horizontal: transfer of genetic material between organisms that are not parent and offspring. Vertical: transfer of genetic material from parent to offspring during reproduction. - Types of horizontal gene transfer: Know what they are and be able to name them if the description is provided - Transformation -- uptake of free DNA from the environment by a competent cell. Example: Griffith's experiment with Streptococcus pneumoniae. - Transduction- transfer of DNA via a bacteriophage (virus that infects bacteria). Types: generalized transduction- random bacterial DNA is packaged. Specialized transduction- specific bacterial DNA segments are transferred. - Conjugation -- direct transfer of DNA between two bacteria through a pilus (conjugation bridge), often involves plasmids, like the F plasmid. - Transfection- introduction of DNA into eukaryotic cells, often using a virus or chemicals. - What is a plasmid?- a circular, double-stranded DNA molecule independent of chromosomal DNA. Often carries genes for survival, such as antibiotic resistance. - Be able to identify and know the functions of the following for **any plasmid map** - Origin of replication- allows plasmid replication. - Promoter- drives transcription of the gene of interest. - Gene of interest- the target gene to be expressed or studied. - Selection gene: Antibiotic resistance gene (know that ampicillin, kanamycin, and chloramphenicol are commonly used antibiotics for bacterial selection)- confers resistance to antibiotics. - Multiple cloning site- region with many restriction enzyme sites for inserting DNA. - What are competent bacteria?- bacteria that can take up foreign DNA, such as plasmids, from their environment. - What chemical is used to make bacteria competent?- Calcium chloride(CaCl2) is commonly used to make bacteria competent. - 2 different methods of transformation: Electroporation and heat shock- Electroporation: brief electric pulses create pores in the bacterial membrane, allowing DNA to enter. High efficiency, especially for large plasmids. Heat shock: sudden temp increases disrupt the membrane, allowing DNA uptake. - Temperature used for heat shock- 42 degrees Celsius for 30-60 seconds. - Differentiate between screening and selection. Which one is better and why?- screening identifies cells with the desired genetic trait based on phenotype, requires additional testing or observation, Example: Blue-white screening using X-gal. Selection: only cells with the desired genetic trait can survive ( antibiotic resistance), more efficient as unwanted cells do not grow. Selection is better for most experiments as it saves time and resources. - Expected growth in different plates - LB- growth of all bacteria (non-selective) - LB + Amp- growth only of bacteria with plasmids carrying the ampicillin resistance gene. Non-transformed bacteria (without plasmid) will not grow. 6. **Plasmid isolation** - Please remember that plasmids are circular DNA - Know the reason for every step of plasmid isolation. Please **do not** memorize the protocol though.  - Culturing: grow bacteria that contain the plasmid in a culture medium, often with an antibiotic to ensure only the desired bacteria grow. - Harvesting: collect the bacterial cells by centrifugation, leaving a pellet. - Resuspending: prepare the cells for lysis by resuspending them in a buffer. - Alkaline lysis: break the cells apart and convert double-stranded DNA to single-stranded DNA - Neutralization: precipitate cellular debris and genomic DNA - Recovery: separate the plasmid DNA from the cleared lysate. - Know the functions of - Resuspension buffer- resuspend the bacterial pellet after centrifugation - Lysis buffer: Function of NaOH and sodium dodecyl sulfate- break open bacterial cells and release their contents, including plasmid DNA. NaOH: raises the pH, denatures chromosomal and plasmid DNA, and disrupts the hydrogen bonds between DNA strands, helps to degrade proteins. SDS- detergent that dissolves lipid membranes and denatures proteins. - Neutralization buffer: Function of potassium acetate- neutralize the alkane lysis reaction and precipitate unwanted components. Potassium: lowers the pH, allowing plasmid DNA to reanneal, precipitates denatured proteins, SDS, and chromosomal DNA - Silica column-bind plasmid DNA selectively while other contaminants pass through. - Wash buffer- remove salts, proteins, and other impurities while leaving plasmid DNA bound to the silica columns. - Elution buffer- release purified plasmid DNA from the silica column. 7. **Spectrophotometric analysis** - Wavelengths we emphasize on during the experiment:- 260, 280, and 230 nm.  - What wavelengths do nucleic acids absorb?- absorption peak: 260 nm - What wavelengths do proteins absorb- absorption peak: 280 nm - What is the desirable ratio for absorbance at 260 nm and 280 nm?- desirable ratio: \~ 1.8 for DNA and \~2.0 for RNA. - What is the desirable ratio for absorbance at 260 nm and 230 nm?- desirable ratio is \~2.0-2.2 - Be able to calculate the concentration of DNA if absorbance is provided- DNA concentration: A260 x dilution factor x conversion factor. Example: The absorbance of a DNA sample at 260 nm is 0.5. The sample was diluted 1:100. Calculate the concentration of the DNA. DNA concentration= 0.5x100x50= 2500 μg/mL. 8. **Restriction digestion** - What are restriction enzymes or restriction endonucleases- proteins that cut DNA at specific sequences called recognition sites. Act as molecular scissors, breaking the phosphodiester bonds of DNA strands. - What organisms make them and why?- bacteria, defense mechanisms against invading viruses(bacteriophages), they cut foreign DNA while protecting their own DNA by methylation of recognition sites. - Identify palindromes that are potential restriction enzyme sites- palindromic sequence: reads the same in the 5' to 3' direction on both strands. Example: 5'-GAATTC-3' 3'-CTTAAG-5'. Recognized by EcoRI. - Determine if an end is blunt or sticky after restriction digestion- Blunt: cuts both DNA strands at the same position. Sticky: cuts at staggered positions, leaving overhangs. - Be able to write the DNA fragments after cutting with a restriction enzyme- Example: sequence- 5'-GAATTCAGCTTGAATTC-3'. Enzyme: EcoRI cuts at GAATTC. Result: Fragments: 5'-GAATTC-3' and 5'AGCTTGAATTC-3'. - Be able to determine number of fragments and size of fragments following restriction digestion for linear and circular DNA.- Linear: number of fragments= number of cuts +1. Circular: number of fragments=number of cuts. Size: subtract fragment lengths from total DNA size based on where cuts occur. - Applications of restriction digestion for DNA fingerprinting- generates unique fragment patterns for individuals based on differences in DNA sequences. Used for: paternity testing, crime scene analysis, genetic diversity studies. - How to calculate volumes of different components for a restriction enzyme reaction.- formula: C1V1=C2V2 for buffer or DNA stock - How to differentiate between cut and uncut samples on a gel.- Uncut: single, undigested band; may appear supercoiled, relaxed, or nicked. Cut: multiple bands depending on fragment number and sizes, bands correspond to expected fragment sizes based on digestion. 9. **Agarose electrophoresis** - What is gel electrophoresis?- used to separate DNA, RNA, or proteins based on size by applying an electric current through a gel matrix. - What type of matrix can be used for electrophoresis?- agarose gel and polyacrylamide gel. - The two functions of the loading dye during gel electrophoresis.- tracking progress and weighing down samples - What is the purpose of 1X TBE or TAE in agarose gel electrophoresis?- maintain pH and prevent nucleic acid degradation - What charge does DNA have? Why?- Negative, due to phosphate backbone. - How would you know when to turn off the electricity while running an agarose gel?- when the tracking dye is about 1-2 cm from the end of the gel to prevent the DNA from running off. - What are the types of stains used for detection of DNA? Explain the principle of how we can detect DNA using these chemicals.- Ethidium bromide(EtBr): intercalates between DNA bases and fluorescence under UV light. Principle: absorbs UV light and emits visible light when bound to DNA. SYBR safe or GelRed: safer alternative to EtBr; bind to DNA and fluorescence under UV or blue light. - Be able to interpret the bands. Size, distance from the wells, number of bands.- size: bands closer to the wells are larger; bands farther away are smaller. Distance: migration distance is inversely proportional to molecular size. Number of bands reflects the number of distinct DNA fragments present. - What is a ladder and what is its purpose?- ladder: a mixture of DNA fragments of known sizes. Purpose: acts as a molecular weight marker to determine the sizes of DNA bands. - How to calculate the amount of agarose needed based on volume and % of gel.- amount of agarose(g)=volume of buffer (mL)x(% of gel/100) - How to calculate volumes for diluting buffers to obtain 1X of TAE of TBE.- C1V1=C2V2. C1: initial concentration of stock buffer, V1: volume of stock buffer required, C2: final concentration, V2: final total volume. - Role of 1X TAE (or TBE), loading dye (buffer, bromophenol blue, glycerol, electricity, SyBr safe (ethidium bromide, gel red), etc. -1X TAE or TBE: provides ions for current flow and maintains pH. Loading dye: buffer- stabilizes pH, bromophenol blue-tracks sample migration, glycerol-makes the sample dense for easy loading. Electricity: drives the negatively charged DNA through the gel matrix toward the positive electrode. Stains: enable visualization of DNA under UV or blue light. 10. **Inducible and constitutive expression and Microscopy** - What is gene expression?- the process by which information from a gene is used to synthesize functional products like proteins or RNA. Gene expression involves transcription (DNARNA) and translation (RNAprotein). - What is the difference between inducible and constitutive expression?- inducible expression means a gene is only expressed when a specific stimulus is present, while constitutive expression means a gene is expressed continuously regardless of environmental conditions. - Why are some genes constitutively expressed?- these genes are essential for survival and are required for basic cellular functions, such as maintaining cell structure, energy metabolism, or protein synthesis. - Why are some genes inducible expressed?- allow organisms to adapt to environmental changes and conserve energy by producing proteins only when needed. - Explain how GFP is expressed in an inducible manner in pGLO- GFP is under the control of the arabinose operon promoter. - What does AraC do in the absence of arabinose?- represses the promoter, preventing GFP transcription. - What does AraC do in the presence of arabinose?- changes conformation, allowing RNA polymerase to bind to the promoter and initiate transcription of GFP. - Does arabinose promote or express GFP expression?- promotes - What are stereoisomers?- molecules with the same chemical formula and connectivity of atoms but differing in spatial arrangement. - Can D- and L-isomers function the same?- No. Biological systems are stereospecific. - Expression of GFP in pGFP and pGlo based on the plates they grew in - LB+Amp- result: bacterial growth, no GFP expression. Reason: ampicillin resistance gene allows growth, but no arabinose is present to induce GFP expression. - LB+Amp+L-Arabinose- Result: bacterial growth with GFP expression (colonies fluorescence under UV light). Reason: arabinose induces GFP expression by activating the arabinose promoter. 11. **Flow Cytometry- starts final exam** - Different parts of instrument (laser, optics, fluidics, detector, electronics)- laser provides the light source to excite fluorophores attached to cells, different lasers emit specific wavelengths. Optics directs and focuses the laser beam and collects the emitted fluorescence and includes filters and mirrors to separate light into specific wavelengths. Fluidics transports cells in a single file through the laser beam using a sheath fluid and ensures consistent measurement of individual cells. Detector captures scattered light (FCS and SSC) and emitted fluorescence and converts light signals into electronic signals for processing. Electronics converts signals from detectors into digital data and allows for data acquisition, compensation, and analysis. - Compensation (what is compensation, when should they be performed)- adjusting for spectral overlap when fluorophores emit light in overlapping wavelengths and ensures accurate measurement of fluorescence in each channel. It should be performed when using multiple fluorophores in a single experiment and must be done before analyzing samples using single-strained controls. - What information does FSC and SSC provide- FSC provides information about cell size and larger cells scatter lighter foreword. SSC provides information about cell complexity or granularity and more granular cells scatter more light to the side. - How to analyze histograms and dot plots (scatter plots) by determining % of cells and whether there is an increase or decrease in the measured parameter. -- Histograms: peaks indicate populations; a shift to the right suggests an increase, while a shift to the left indicates a decrease in fluorescence or other parameters. Dot plots quadrants can be used to identify populations. Determining % of cells: calculate the percentage by dividing the number of gated cells by the total number of cells. Increase or decrease look for shifts or changes in the population density in histograms or dot plots, compare treated vs. control samples to observe trends in the measured parameter. 12. **Mendelian Genetics** - How to perform Mendel's monohybrid and dihybrid crosses using Punnet square - Determine the phenotypes and genotypes of F1 and F2 generation after performing monohybrid and dihybrid crosses- In a monohybrid cross, the F1 generation will all show the dominant phenotype and be heterozygous, while the F2 generation will show a 3:1 phenotypic ration with genotypes in a 1:2:1 ratio. In a dihybrid cross, the F1 generation will all show the dominant phenotype for both traits and be heterozygous for both genes, while the F2 generation will show a 9:3:3:1 phenotypic ration, being the different combinations of dominant and recessive traits for both genes involved. - Remember the ratio of phenotype and genotype in Mendel's monohybrid cross and ratio of phenotypes in Mendel's dihybrid cross when heterozygous parents are involved- monohybrid cross- 3:1 ratio. Dihybrid cross: 9:3:3:1 ratio. - If genotypes of parent are provided, determine the genotype and phenotype of progeny- 1) name the parental genotypes. 2) set up the Punnett square. 3) fill in the alleles. 4) combine alleles. 5) interpret the results. - Predict the phenotype from the genotype - Tt x tt= Tt tt Tt tt = 50% Tt (heterozygous tall, 50% tt (homozygous short). 50% tall, 50% short - Describe Mendel's principles of heredity- 1) Principle of segregation: when gametes are formed, the pairs of hereditary factors (genes) become separated, so that each sex cell (egg/sperm) receives only one kind of gene. 2) Concept of dominance: one allele masked another; one allele was dominant over the other in the F1 generation. 3) Principle of independent assortment: two traits separate independently of each other or the alleles of a gene segregate independently of other genes. - Determine if an organism is homozygous or heterozygous from the genotype- If alleles for a trait are identical (ex: AA or aa) it is homozygous. If alleles for a trait are different (ex: Aa) it is heterozygous. - Determine if a trait is dominant or recessive based on genotype and phenotype- If only one copy of a specific allele is needed to express the trait, its dominant; if an individual needs two copies of the same allele to show the trait, its recessive; typically, dominant alleles are represented by capital letters. While recessive alleles are represented by lowercase letters. - Perform Chi-square analysis if data is given (I will provide the formula and the table) - How to interpret statistical significance based on P-value- a result is considered statistically significant if the p-value is less than a predetermined threshold typically set at 0.05 13. **DNA extraction** - Know what genes, chromosomes, and genomes are.- Genes: segments of DNA that code for proteins or functional RNA molecules. Act as the basic units of heredity. Chromosomes: structures made of tightly packed DNA and associated proteins. Humans have 46 chromosomes (23 pairs) in each somatic cell. Genome: the complete set of genetic material in an organism. Includes all genes and non-coding regions of DNA. - What are exons and introns?- Exons: coding regions of a gene that are expressed as proteins or functional RNA. Introns: non-coding regions of a gene that are removed during RNA splicing. - On what chromosome is the PV92 locus?- Chromosome 16 - Applications of DNA extraction.- Research: genetic studies, gene cloning, and sequencing. Medical: diagnosing genetic disorders, forensic analysis, and pharmacogenomics. Biotechnology: genetically modified organisms (GMOs), DNA fingerprinting. - Why was saline used and not water during the cell collection step?- Saline: isotonic solution that prevents cell lysis during collection, maintains the integrity of cells by matching the ionic strength of the body. Water: hypotonic and can cause cells to swell and burst, releasing contents prematurely. - How do we lyse (make them die) the cheek cells to release the DNA?- Methods: heat treatment- denatures proteins and breaks cell membranes. Detergents- disrupt lipid membranes to release DNA. Physical shearing: sometimes used to assist the process. - Role & function of InstaGene matrix in PV92 PCR Kit.- InstaGene matrix: a chelating agent that binds divalent cations. Functions: Protects DNA- chelates magnesium ions, which are cofactors for DNases. Purifies DNA: removes impurities and ions that could inhibit downstream applications like PCR. 14. **PCR** - Full name of PCR: Polymerase Chain Reaction. - Who invented the technique?- Kary Mullis in 1983. - Purpose of PCR.- amplify specific DNA sequences, enabling the generation of millions of copies from a small DNA sample. - Components needed for PCR: - DNA of interest- the target DNA to be amplified. - Heat-stable DNA polymerase- Taq polymerase, remains functional at high temps required for denaturation. - All 4 nucleotide triphosphates- dATP,dTTP,dCTP,dGTP provide the building blocks for DNA synthesis - Buffers **-\>** conditions that allow reaction to happen- maintain the optimal conditions for the polymerase to function. - Two Primers (forward and reverse): Know how to design them- Forward: binds to the 5' end of the sense strand. Reverse: binds to the 5' end of the antisense strand. - Thin-walled tubes for easier heat transfer- facilitates rapid heat transfer during cycling - Thermal cycler- instrument that changes temps for each step of the PCR cycle. - Steps of PCR: Know all 3 steps. Know the temperature, time, and purpose of each step.- 1) Denaturation: temp: 95C, time: 30 sec -1 min. purpose: break hydrogen bonds between DNA strands to separate them. 2) Annealing: temp: 5 C below the melting temp of the primers. Time: 30 sec. Purpose: allow primers to bind to the complementary sequences on the DNA strands. 3)Extension: Temp: 72C Time: dependent on fragment size, generally 1 min per 1 kb. Purpose: polymerase synthesizes new DNA strands by extending primers. - How to determine the temperature of annealing step from Tm of primers.- calculated using Tm=4(G+C)+2(A+T), where G,C,A,T are the counts of respective nucleotides in the primer. - How to determine time for extension step?- Ta=Tm-5C, Tm= melting temp of the primers, - Why do we need heat stable DNA polymerase?- it withstands high temps during denaturation without denaturing, ensuring efficiency in repeated cycles. - Controls needed for PCR: Negative control, positive controls- Negative: reaction without template DNA to check for contamination. Positive: reaction with a known template to confirm the reaction conditions and reagents are working. - Find how many molecules after PCR cycles - N~t~ = N~0~ \* 2^n^ - N~0~ is number of molecules - n is number of cycles - DNA of interest amplified by a power of for each PCR cycle - Determine patterns for individuals homozygous for presence or absence of PV92 or heterozygous observed in agarose gel electrophoresis.- Homozygous presence(++)- one bright band at the amplified region. Homozygous absence(\--)- no band at the amplified region. Heterozygous(+-)- two bands, 0ne for each allele. 15. **Population Genetics** - - - - - - - - - - - - - - - - - - - - - - - - - - - -