BCH 301 Lab Exam Final List PDF

BCH 301 Lab Exam: Final List LAB 1 ● Pipetting List of tips & points for using a pipette: 1. 2. 3. 4. 5. 6. 7. 8. Prewet the pipette tip - not doing this can cause lower delivery volumes Work at temperature equilibrium - this minimizes variation of volume Make sure the tip is dry before you put...

BCH 301 Lab Exam: Final List LAB 1 ● Pipetting List of tips & points for using a pipette: 1. 2. 3. 4. 5. 6. 7. 8. Prewet the pipette tip - not doing this can cause lower delivery volumes Work at temperature equilibrium - this minimizes variation of volume Make sure the tip is dry before you put it in Use standard mode pipetting Pull the tip straight out Minimize handling of the tip Immerse the tip to a proper depth Use the correct size and brand of tip First stop → insert → upper stop → lift out → place in desired tube → first stop → second stop → all the way up to upper stop. ● Lab safety LAB 1 ● Safety List of general main safety points: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Do not perform any experiments in the laboratory unsupervised Wear your goggles Clothes must cover all of your body No open-toed shoes Hair must be tied back Wear gloves when handling chemicals Keep a clean workspace No eating, drinking, smoking, chewing gum Wash hands before leaving the lab Don’t bring chemicals out of the lab LAB 2 & 3 ● Titration curves: pKa of weak acids, weak acid/base dissociation constants - pH reflects, on a logarithmic scale, the concentration of hydrogen ions - Acids partially ionize & donate hydrogen … lowering the pH of the solution. Bases accept this hydrogen ion and increase the pH This process is characteristic of each unique acid or base and is expressed as an acid dissociation constant & - The pkA expresses the strength of a weak acid or base - The stronger the acid the smaller its pKa The stronger the base the larger the pKa (its conjugate acid ^^) The pKa is the midpoint of the titration curve - Buffer: solution that can resist pH change upon addition of an acid or base - it neutralizes small amounts and creates a stable pH. LAB 2 & 3 Post lab Q’s that are relevant: 1. ​A 25.0 mL sample of 0.30 M formic acid is titrated with 0.01 M magnesium hydroxide. What is the pH of the solution after 10 mL of Mg(OH)2 has been added? Please show all calculations. (Ka for formic acid = 1.8 x 10-4M) 2. A buffer consisting of H2PO4- and HPO42- helps control the pH of physiological fluids. Many carbonated drinks use the same buffer system. What is the pH of a soft drink in which the major buffer components are 5 g of NaH2PO4 and 3 g of Na2HPO4 per 250. mL of solution? Examples of titration curves: ● ● ● ● ● ● Beer-Lambert Law Working with Proteins: isolation, chromatography, electrophoresis, activity Working with carbohydrates and lipids (general) PCR Sanger Method Centrifugation The lab itself; We used an acid (HCL) to bring the pH down then continuously added increments of a base (NaOH) until we reached a pH of 12. Data was graphed & buffer regions were identified (+/- 1 of the pKa). Since the pKa’s are unique to an amino acid, the unknown was determined. LAB 4 ➔ 3.1 Beer-Lambert Law - Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution. The fraction of light absorbed is related to the thickness of the absorbing layer & the concentration of what is absorbing…this is known as the Lambert-Beer Law Io = intensity of the incident light, I = intensity of the transmitted light, Io/I = the ratio (the inverse of the ratio in the equation) is the transmittance, E = molar extinction coefficient (in units of liters per mole-centimeter), c = concentration of the absorbing species (in moles per liter), L = path length of the light-absorbing sample (in centimeters). **The Lambert-Beer law assumes that the incident light is parallel and monochromatic (of a single wavelength) and that the solvent and solute molecules are randomly oriented. The expression log is called the absorbance, designated A. **Proteins can be assayed easily with a spectrophotometer → measures the UV region, for example Tryptophan & Tyrosine absorb light at 280 nm. When you can't come upon a spectrophotometer to use UV absorbance, you use the Bradford method → uses Coomassie Brilliant Blue G-250 (neg charge). Goes from red → blue when it binds to a positive charge on a protein 1. What is the theoretical absorbance at 340 nm of a 0.01 M solution of NADH, assuming a pathlength of 1 cm? 2. What dilution would be necessary to get the absorbance from question 1 down to 3.1? (_____mL of 0.1 M NADH to _____mL H2O) The lab itself; a spectrophotometer was used to calculate the concentrations using beer's law. A simple protein assay was done and standard curves were used to determine the protein concentration of the unknown. LAB 5 ➔ 3.3 Working with Proteins (All)/ Working with Proteins: isolation, chromatography, electrophoresis, activity - - - - - Column chromatography - Step 1: buffered solution / mobile phase runs through the solid phase (gel) - Step 2: now the solution with the protein migrates again Ion-exchange chromatography - Separates based on sign & magnitude of charge - Two solid phases → 1. Cation exchange & 2. anion exchange - Everything passes through except the protein of interest - The packed beads have the opposite charge of the protein of interest - (-) charged beads = cation exchange , elutes (-) protein first Size - exclusion chromatography - microscopic beads which contain tiny holes are packed into a column - Separates based on size - Large proteins elute first affinity chromatography - Separates based on binding affinity - Stationary / solid phase is chosen based on which you want to elute first - Higher affinity will elute second - everything passes through except the protein of interest, which binds to the antibody and is retained on the solid support. High - performance liquid chromatography - Uses high-pressure pumps to move down a column ➔ 3.4 Mass Spec, Synthesis - Mass Spectrometry : measures molecular mass - Can sequence a short amino acid sequence - Can document the proteome - Steps: 1. Ionize analytes in a vacuum 2. Introduce charged molecules to electric or magnetic field 3. The charged molecules move through field 4. Deduce mass of analyte - - Tandem mass spec: - Two masses filters in tandem - 1. Sorts peptides produced by oritease - 2. Measures m/z ratios of charged fragments Synthesise; - Use a protecting group - the merifield method The lab itself; Used size exclusion chromatography to separate molecules based on size. Hemoglobin and vitamin B12 were used. Hemoglobin is brown (heavier) and Vitamin B12 is pink (lighter). Hemoglobin elutes first due to size LAB 6; Part 1 ➔ 3.3 Working with Proteins /Working with Proteins: isolation, chromatography, electrophoresis, activity ➔ Electrophoresis: visualize and characterize purified proteins ◆ can be used to estimate: number of different proteins in a mixture – degree of purity isoelectric point approximate molecular weight ➔ uses cross-linked polymer polyacrylamide gels ➔ proteins migrate based on charge-to- mass ratio ➔ visualization = Coomassie blue dye binds to proteins ➔ SDS Electrophoresis: ◆ SDS is a detergent that binds and partially unfolded proteins ◆ Gives all proteins a similar charge-to-mass ratio ◆ When together proteins are separated by molecular weight ◆ *smaller proteins move down faster ➔ Two - dimensional electrophoresis … More sensitive Step 1: use isoelectric focusing to determine the PI of a protein Step 2: electrophoresis The lab itself; Began by extracting proteins from muscle tissue, unfolded them, and denatured them. A negative charge was given using a sample buffer, flicking the tube, and heating the protein. Gel electrophoresis was then used to separate the proteins by molecular weight. LAB 6; Part 2 Does species E share the five proteins that the common ancestor of species B, C, and D had? No. Does species E share more proteins with B, C, and D than A? Yes. Therefore, species E gets its own branch in between the D and A branches to indicate that it has more shared characteristics with B, C, and D than A, but fewer shared characteristics with B and C than D. The lab itself: observed the gel by measuring the distance the proteins migrated from the wells to the base. Only proteins less than 40 kD are measured. This length was plotted vs MW. Similarities of these points were drawn and an HR table was created. The more commonality the closer related the fish. A phylo tree was created (see above.) LAB 6; Part 3 ➔ 5.2 Western Blot - Western Blotting 1. antibody is attached to a reagent that makes it easy to detect / aka “blotting” a. To probe the samples with the myosin-specific antibody, proteins must first be transferred or "blotted" from within the gel onto the surface of a membrane. 2. When the antibody binds the target protein, the label reveals the presence of the protein in a solution or its location in a gel, or even in a living cell. 3. In one application of this technique, proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. 4. After washing, the membrane is treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. 5. A colored precipitate forms only along the band containing the protein of interest. ​ The lab itself; Western blotting was completed, using antibodies so that in part 4 we could identify myosin light chain proteins in the fish muscle extract. LAB 6; Part 4 - Immunodetection Steps: 1. Primary antibody is added to the blot and incubated to allow the antibody to bind to the myosin protein on the membrane. 2. The unbound antibody is then washed away. 3. The primary antibody provided is a monoclonal mouse anti-myosin light chain antibody. 4. Secondary antibody is added to the blot and incubated to allow the secondary antibody to bind to the primary antibody. 5. The unbound secondary antibody is then washed away. 6. The secondary antibody is a polyclonal goat anti-mouse antibody conjugated to HRP. 7. The secondary antibody was produced by injecting goats with primary mouse antibodies. 8. HRP is the enzyme that catalyzes oxidation of the colorimetric substrate so the protein of interest can be identified. 9. Colorimetric (color-producing) enzyme substrate is added to the membrane and incubated to allow color to develop. 10. Purple/gray bands will develop on the membrane exactly where the myosin protein bands are located. 11. When oxidized by HRP in the presence of hydrogen peroxide, this colorless solution forms a purple/gray precipitate that binds to the membrane at the antigen location. ➔ Troubleshooting: ➔ ➔ ➔ ➔ ➔ ➔ ➔ ➔ Extra bands: Too large of samples in initial tissue extraction step Vertical lines: accidental transfer of muscle protein Halo Bands: overloading, too large of sample in initial tissue extraction No prestained proteins / bands appear on the gel: transfer of gel may have been done wrong, incorrect set up of power supply, the sandwich was set up incorrectly Only prestained standards are present: primary & secondary antibodies have been mixed up or accidentally omitted - reagent may have been over exposed to light Bands have small white circles or holes in them: air bubbles Bands are smiling or frowning: issue with the gel or gel running buffer. Many reasons within this, one is that the buffer level in the inner chamber may have been too low due to a leak. Bands are pale: blotting issues - poor construction of the blot. The lab itself: In step 3, antibodies were used to detect a specific protein. In this lab, the blot has finished and the gel for analysis is prepared for antigens / immunodetection. LAB 7 ➔ 8.3 Melting Curves ◆ We can estimate the C-G & T-A ratio by solving for the denaturation temperature (tm) → temp at which ½ of DNA is single stranded due to increased temp. ◆ **an increase of C-G content will increase tm due to the increased number of hydrogen bonds when compared to T-A ◆ Denaturation starts at A-T , microscopically you these can be identified as bubbles ● This is b/c they are easier to separate than G-T due to less H bonds, so there is a lower melting point and a faster denaturation ➔ PCR; method of amplifying DNA segments of interest ◆ Relies on DNA polymerase → adds nucleotides to the 3’ ends (aka the primers) ◆ PCR can be used to detect & amplify just one DNA molecule in any sample type ◆ Components; ● DNA sample ● Pair of synthetic oligonucleotide primers ● Pool of dNTPs ● DNA polymerase (Taq) ◆ Steps; ● Reaction is heated to denature the DNA ● Mixture is cooled so that the primers can anneal to the DNA before the DNA itself does ○ A high concentration of primers prevents DNA from self annealing ● Primed segment is then replicated selectivity by DNA polymerase ➔ Sanger Method; dideoxy chain-termination sequencing used to determine DNA sequences ◆ ddNTPs interrupt synthesis at one nucleotide ● They bind to template strands but lack s 3’-hydroxy group to add to the next nucleotide so the strand cannot be extended further ◆ Makes use of DNA polymerase + primer ◆ Steps; Identifying C residues ● Add ddCTP ● When DNA polymerase encounters a G in the template strand, dC is added and the reaction continues ● But, when ddC is added the strand is terminated (this happens at the same time as the previous step) ● The resulting solution now has a mixture of fragments ending with ddC ➔ DNA fingerprinting & sequencing ◆ Application → paternity testing ◆ STR’s → short tandem repeats, are simple regions that repeat over & over again ● The number of repeats is what gives them variability ◆ Restriction endonuclease can cleave DNA into smaller pieces in a predictable manner, around 4-8 base pairs long @ the recognition site ● Two ways to cut ○ 1. Blunt / straight ○ 2. Staggered / sticky *The steps to fingerprinting; 1. Isolate DNA → hair, skin, saliva.. 2. Cut using restriction enzymes 3. Gel electrophoresis is performed on the fragmented sample a. DNA molecules have a negative net charge. b. The gel is placed so that DNA samples are at the side of the chamber with the negative electrode. c. current is applied & negatively-charged DNA fragments are attracted to the positive electrode on the other side of the chamber. d. Because the gel containing the DNA samples has microscopic pores, smaller DNA fragments are able to weave their way through the gel more readily than larger DNA fragments. 4. The result of the gel; smaller fragments have moved farther down the gel 5. Fragments are denatured 6. Southern blotting is conducted 7. The blotted DNA is then exposed to radioactive probes which attach to specific DNA sequences 8. An x-ray film is used and the radioactive probes will expose themselves → dark bands will appear where the probed sequences of DNA are present. The lab itself; An agarose gel electrophoresis was performed on four different DNA samples, the mother, child, and two possible fathers. The blotted DNA was then analyzed to determine who the father was. Miscellaneous Lab Tech From the Textbook ➔ 4.5 XRC, NMR ◆ X-ray crystallography: produces an electron density map from protein crystals ● Patterns of x-ray diffractions are collected & an image is created mathematically ● Dependent on the degree of structural order - Provides little information about the molecular movement of proteins Representative of a functional conformation ➔ NMR; determines distance between protein atoms ◆ captures dynamics of protein structure: ● conformational changes ● protein folding ● interactions with other molecules ◆ Measures nuclear spin of H, C, N, F, and P ● The nuclear spin generates a magnetic dipole ◆ This magnetic field causes the magnetic dipoles to align in the field in one of two orientations: parallel (low energy) or antiparallel (high energy) ◆ pulse of electromagnetic energy generates an NMR spectrum ● Two types of NMR techniques: 1. NOESY = measures distance - dependent coupling of nuclear spins in nearby atoms through space 2. TOCSY = measuring coupling of nuclear spins in atoms connected by covalent bonds. ➔ 7.5 Working with Carbohydrates (All) Methods of purification: ◆ ◆ ◆ ◆ Fractional Precipitation by solvents - “salting out” Ion-exchange chromatography Size exclusion chromatography Affinity chromatography → using highly purified lectins attached to insoluble support Methods of Carbohydrate Analysis (more complex than proteins & nucleic acids) ➔ We can determine sequence, configuration at anomeric and other carbons, and positions of glycosidic bonds: ◆ traditional chemical and enzymatic approaches ◆ mass spectrometry ◆ high-resolution NMR spectroscopy Methods of Synthesising ➔ solid-phase oligosaccharide synthesis: ◆ based on the same principles as peptide synthesis ◆ yields defined oligosaccharides ◆ useful in exploring lectin-oligosaccharide interactions ➔ 9.1 DNA cloning The process; 1. obtain the DNA segment to be cloned a. Restriction endonuclease = precise scissors that are used to cleave DNA into smaller specific fragments 2. select a small molecule of DNA capable of autonomous replication a. Cloning vectors are used to generate recombinant DNA 3. join the two DNA fragments covalently a. DNA ligase links the cloning vectors to DNA fragment to be cloned 4. move the recombinant DNA from the test tube to the host organism a. E. Coli/BAC, plasmids, yeast/YAC 5. select or identify host cells that contain the recombinant DNA a. Cloning vectors have features that allow host cells to survive in… an environment in which cells lacking the vector would die – Cells containing vector are thus “selectable” ➔ 9.2 Determining a protein's function Determining a protein's function by telling where and when it is 1. RNA-Seq ➔ method that determines the RNAs that are transcribed from a genome under a given set of conditions ➔ Usually used alongside mass spec & provides info into how proteins are modified 2. Immunofluorescence ➔ alternative approach for visualizing the endogenous protein that involves fixation (and death) of the cell Determining how a protein interacts to suggest its function 1. 2. 3. 4. TAP (tandem Affinity Purification) Fuses the gene that encodes for a protein of interest with a gene for an epitope tag Precipitate the protein product by complexing it with the antibody that binds to the epitope ◆ ^^ immunoprecipitation → helps identify associated proteins ◆ Proteins that bind to the tagged protein will also precipitate Now you can do affinity chromatography to separate the precipitated proteins ◆ crude extract of cells that express a tagged protein is added to a column with immobilized antibodies ◆ the tagged protein binds to the antibody, ● proteins that interact with the tagged protein are sometimes also retained on the column. ◆ The connection between the protein and the tag is cleaved with a specific protease. The proteins eluted from the column are identified by mass spec ➔ CRISPR/Cas systems ; Determining deleting or altering a protein to determine its function Two main terms: CRISPR sequences = regularly spaced short repeats in bacterial genome Cas Proteins = nucleases / components of immune system that have evolved to allow bacteria to survive infection by bacteriophages ^^these both are components of a bacterial immune system Breaking it down; Crispr/Cas Complex Components = the complex binds and destroys invading bacteriophage DNA by the Cas protein nuclease 1. Guided RNAs 2. Trans-activating CRISPR RNA (tracrRNA) 3. 1+ Cas proteins Current CRISPR tech Only requires two components: 1. 1 cas protein to cleave DNA (Cas9) - Has two separate nuclease domains - Each domain cleaves one strand of dna - Inactivating one creates enzyme that cleaves just one strand 2. Single guide RNA (sgRNA) - Made of gRNA and tracrRNA fused into 1 RNA - The sequence can be altered to target any genomic sequence This is required to pair with the target DNA sequence and to activate the nuclease domains Additional points to make ➔ If mutation is required, it can be introduced by recombination when DNA fragment enters cell with CRISPR/Cas9 plasmids ➔ Can be combined with other approached such as RNA- Seq for additional information - Research, medicine, genetic screening, and more ➔ Plasmids expressing required protein & RNA components can be introduced to cells by electroporation. ➔ 10.4 Working with Lipids (All) Generals of Separating lipids → in general lipids are separated by differences in polarity or solubility in nonpolar solvents ➔ Neutral lipids ◆ Extracted with ethyl ether, chloroform, or benzene ◆ These solvents do not allow for clustering due to the hydrophobic natures of lipids ➔ Membrane lipids ◆ Extracted by ethanol or methanol ● Reduces the hydrophobic interactions ● Weakens the hydrogen bonds & electrostatic interactions that bind membrane lipids to membrane proteins * a commonly used extractant is a mixture of chloroform, methanol, and water (1:2:.08) Methods of separating lipids ➔ Adsorption Chromatography → Separates Lipids of Different Polarity ◆ Silica is a polar material used to separate the mixtures ◆ Order of elution … (1) neutral lipid (2) less polar (3) polar ➔ Gas Chromatography → Resolves Mixtures of Volatile Lipid Derivatives ◆ Turns lipids to gas so they can be carried by a stream of inert gas (He) ◆ The lower boiling point will elute first Determining Lipid structure ➔ Specific Hydrolysis: ◆ all ester-linked fatty acids are cleaved/cut/broken off by mild acid or alkaline treatment ◆ harsher hydrolysis conditions cleaved/cut/broken off amide-bound fatty acids from sphingolipids ◆ phospholipases specific for one of the bonds in a phospholipid generate simpler compounds ➔ Mass Spectrometry ◆ allows the analysis of crude mixtures of lipids without prefractionation ◆ can determine the length of a hydrocarbon chain or positions of double bonds but not cis & trans