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BIOB12 - Cell and Molecular Biology Laboratory Lecture notes. Lecture 1 Dilutions •

The ability to prepare dilutions from previously made solutions is a critical skill in biosciences.

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For example, –

to make dilutions of a standard mixture to conduct an assay

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make serial dilutions to find the right antibody titer or enzyme activity level or to dilute live cells.

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Knowing how to dilute solutions allows you to make working solutions from stock solutions, saving a lot of time

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Most of the time you will dilute from a known higher concentration to a new, more dilute concentration by adding water or a buffer solution.

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When you add only water to a solution: – Concentration will change – Volume will change – The total amount of substance does not change.

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Given by the equation: – CxV=A

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C = concentration of a substance, V = volume of the solution, and A = the total amount of substance in the solution.

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Always move from high concentration to low concentration.

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The total amount of substances in the solution will never change when diluting

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Identify the type of problem (The first thing you do is determine what kind of situation you are in?):

– dilute the whole thing to desired final concentration – dilute to desired final concentration and volume – prepare a dilution from a stock solution •

set up the problem C1(S)V 1(S) = C2(F)V2(F)

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determine which variable is the unknown

Dilute a Known Starting Volume

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After determining the final volume that is created, the difference between the final and initial volumes are take to see how much volume was added to reach the final

Example for Performing a Dilution •

Dilute 10 ml 0.125M NaCl to 0.025M V1 = 10 ml = 0.01 liter C1 = 0.125 moles/liter C2 = is to be 0.025 moles/liter Solve for V2 V2 = V1C1/C2 = (0.01L)(0.125M)/0.025M = 0.05L adjust to 0.05L (50 ml)

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You are going from a higher concentration to a lower concentration therefore, you determine the final volume and then the Q.s or the amount of volume added

Dilute to a Specific Volume and Concentration

Dilution Example: Dilute to a Specific volume and Concentration •

Prepare 500 µl 3 mg/ml lysozyme where the stock concentration is 25 mg/ml C1 = 25 mg/ml C2 = 3 mg/ml V2 = is to be 500 µl = 0.5 ml Solve for V1 V1 = V2C2/C1 = (0.5 ml)(3 mg/ml)/25 mg/ml = 0.06 ml = 60 µl

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To make 500µl bring 60 µl to final volume of 500 µl by adding H2O

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This is a lysozyme solution – creating an enzyme solution.

Percent Solutions •

Percent (%) solutions: The amount of a substance (either solid or liquid) is expressed as a percentage of the total solution volume (in rare cases, it is expressed as a percentage of total solution weight) – A 1% solution means that there is 1g or 1 ml of the substance in 100 ml of the final solution (or 100 g if using weight)

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Examples: – 1% NaCl (w/v) = 1 g of NaCl in the final volume of 100 ml. – 10% glycerol (v/v) = 10 ml glycerol in the final volume of 100 ml.

Stock Solutions •

Working solution = diluted stock solution (e.g. 1X working solution)

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Stock solution = stored concentrate (e.g. 10X stock solution)

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Dilution factor = ratio of stock concentration to working concentration

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Example: – Stock concentration = 500 mM – Working concentration = 50 mM – Dilution factor = 10X

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Stock solution is the concentrated version of the working solution.

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Create a stock solution to take from instead of creating a brand new solution every time you run an experiment.

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If you have a stock solution and working solution, you always need to dilute the stock solution to the right concentration otherwise you will end up adding more concentrated solution to the experiment than usual therefore your experiment will not work.

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Stock solutions can be used to prepare different working solutions that share the same components but in different proportions.

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Stock solutions are better for making very dilute buffer solutions because we can pipet a small liquid volume more accurately than we can weigh an amount of dry chemical.

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A stock solution can be used as a component of a more complex solution

Make 5mM Sodium Phosphate buffer solution from a 100 mM stock solution

Make 5mM Phosphate Buffered Saline (with 0.85% NaCl) •

To make a working solution with sodium chloride stir in 8.5 g NaCl, add 50 ml 20x sodium phosphate stock, and adjust to 1 liter

Lecture 2 – Spectrophotometry; Protein and DNA Quantification Assays; Making Bacterial Growth media Quantification and Assays •

Most laboratory procedures require one to know the concentration of a substance such as DNA, protein, functional enzymes, or other molecules in a volume of solution.

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You can’t rely on guesswork, and can’t count molecules directly.

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Therefore, need an direct or indirect detection method that is sensitive to a specific substance.

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Must identify a physical property that changes predictably with the concentration of the substance that is to be assayed. – This property must be something that can be seen and measured, either directly or using instrumentation, such as colour, radioactive emission, or sound. – The property must be quantifiable. Finally, must be able to calibrate a detection method.

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Characteristics of an Assay –

A physical property that can be observed and measured

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Sensitive to changes in concentration

– Sensitive to a specific substance – Changes are predictable and quantifiable – Capable of calibration – You can check the absences or amount od substance in a sample

– We are able to calibrate our instruments to measure our known amounts so that we are able to calculate and practically figure out the unknown. Principles of Spectrophotometry •

Biological chemicals absorb light in the UV-Vis range due to resonance of bonds

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Need relationship between given compound and its absorption characteristics to detect and measure that compound in a given solution

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Direct or indirect spectrophotometry

Lambert’s Law

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Beer-Lambert Law: absorbance is directly proportional to solute concentration and to the light path length

Beer-Lambert Law: A= cl •

Extinction coefficient, , is related to electronic environment of light-absorbing unit – Anything that affects electronic environment, changes : pH, ionic strength, solvent – Defined for a particular solvent at a particular  (usually independent of concentration)

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‘A’ indicates the Absorbance of the sample (generally written as A, Abs or OD (optical density) followed by wavelength written as subscript e.g. A280 / OD280)

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C is the concentration and ‘l’ is the pathlength (generally 1 cm)

Spectrophotometer •

Absorbance and Transmittance have an inverse relationship.

Instrument Limitations

Direct Spectrophotometry •

Measure the values directly (i.e. without using secondary reagents) using the physical characteristics of the material being tested e.g. DNA

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Nucleotide bases absorb at 260 nm: – For pure nucleic acid:

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1 OD260 = 50 µg/ml of DNA

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If measuring RNA then 40 µg/ml of RNA

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If pure single-stranded DNA has an absorbance of 1 at 260 nm, then it contains approximately 33 µg/mL of DNA.

Aromatic amino acids tryptophan (Trp) / tyrosine (Tyr) / phenylalanine (Phe) absorb at 280 nm – These amino acids have different extinction coefficients  makes it difficult to relate absorbance to concentration (e.g. M of trp = 5000; tyr = 1400; phe = 190) – Not all proteins contain same # of Trp / tyr / phe – approx. 0.4-1.5 mg/ml of protein per 1 OD280 •

For example, 1 mg/mL of bovine serum albumin is reported to have an A280 value of 0.7. Antibodies (which are a type of protein) at a concentration of 1 mg/mL are reported to have an A 280 between 1.2 and 1.35.

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A very rough rule is that if a sample containing pure protein has an absorbance of 1 at 280 nm, then it contains approximately 1 mg/ml of protein.

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Peptide bonds absorb 190-230 nm but can be affected by buffers, oxygen

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Concentration of protein = ~ 1 mg/mL X the absorbance at 280 nm e.g. if a protein sample has an abs of 0.5, then the concentration of protein in that sample is 0.5 mg/ml

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If using 205nm, then concentration of protein = ~ 15 mg/mL X the absorbance at 205 nm – Notes: Values for proteins vary. The rule that 1 mg/mL of protein has an absorbance of 1 is approximate. Tris and other common solvents also absorb light at 205 nm. For this reason, 280 nm is far more commonly used for protein measurement

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It is possible to use UV spectrophotometry to estimate the purity of a solution of nucleic acids. – involves measuring the absorbance of the solution at two wavelengths, usually 260 nm and 280 nm, and calculating the ratio of the two absorbance values:

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An A260/A280 ratio of 2.0 is characteristic of pure RNA

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An A260/A280 of 1.8 is characteristic of pure DNA

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A260/A280 ratio of about 0.6 is characteristic of pure protein

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Therefore, a ratio of 1.8 - 2.0 is desired when purifying nucleic acids. (Note that this method does not actually distinguish DNA and RNA from one another.)

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A ratio less than 1.7 means there is probably a contaminant in the solution, typically either protein or phenol.

Turbidity •

Turbidity causes an apparent increase in the absorbance of a sample leading to incorrect readings. To compensate for slight turbidity, a background correction can be used. Proteins and nucleic acids do not absorb at 320 nm. Therefore, if a sample absorbs at 320 nm, the absorbance is due to turbidity. The absorbance at 320 can be subtracted from the readings at 260 nm and 280 nm.

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Therefore: – Concentration of double-stranded DNA ~ 50 µg/mL x (A260 - A320) – Concentration of single-stranded DNA ~ 33 µg/mL x (A260 - A320) – Concentration of RNA ~ 40 µg/mL x (A260 - A320) – Concentration of protein ~1 mg/ml x (A280 – A320)

Using Spectrophotometry to Determine form of DNA •

The relative amount of single- or double-stranded DNA in solution can be experimentally determined using spectrophotometry to measure ultraviolet light absorbance at a wavelength of 260 nm (OD260).

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The aromatic bases in DNA are less accessible to ultraviolet light in the double-stranded, compared to single-stranded form, which creates a measurable difference in the observed OD260.

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Using this empirical difference in absorbance, it is possible to observe the effect of temperature on DNA structure by monitoring OD260 over a range of temperature

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The temperature at which 50% of the DNA is denatured is called the Tm or Melting temperature.

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UV compatible cuvette is used – Quartz – UV compatible plastic – Normal plastic cuvette will absorb / quench

Indirect Spectrophotometry •

Compound of interest is colorless

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Can react compound with a secondary reagent to produce a color product

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Measure Abs of colored product -- directly related to [original compound]

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Color reagent must be in EXCESS for this to be true

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Compound of interest always consumed

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Only interested in the reacted compound

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The compound of interest reacts with the reagent completely therefore in an indirect setting the compound s completely consumed .\

Types of Indirect measurement of Protein •

Bradford

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Lowry

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Biuret

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BCA (Bicinchoninic Acid) assay

Bradford Assay/Dye Binding •

Coomassie Blue G-250 dye

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Hydrophobic (tryptophan, tyrosine, histidine and phenylalanine) and electrostatic (arginine, Lysine) amino acids – the assay primarily responds to arginine (eight times as much as the other amino acids) so if you have an arginine rich protein, you may need to find a standard that is arginine rich as well.

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After reacting with protein, the dye shifts Amax from 465nm (brown) nm to 595nm (blue)

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Inhibited by detergents

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Read A595

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Prepare standard curve and calculate [protein]

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Detection Limitations – 1-20 µg (micro assay) 20-200 µg (macro assay) approx.

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Characteristics – Fast (15 min: most other assays take an hour) and inexpensive – Very sensitive – Compatible with a wide range of substances – Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin) – Dye reagent in complex is stable for approximately one hour

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General Considerations – The dye binds to quartz cuvettes so it is usually better to use glass or plastic cuvettes – The dye reagent is usually more convenient to purchase than to make, due to the use of phosphoric acid

Preparation of Bacterial Media •

Media is the nutrient source to grow bacteria

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The media you would have made in the lab is called LB (Lysogeny broth) - a nutrient rich media at pH ~ 7

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This media is sterilized in an autoclave before use  heats the media at high temperature (121oC) under pressure (15 lbs/in2)

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If necessary can add an antibiotic to the media  prevents unwanted bacteria from growing

Making LB broth and LB/Agar media

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Dissolve LB in water while stirring to make the broth

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LB/Agar will not dissolve as agar needs to be heated to dissolve

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Autoclave to sterilize - heat at high temperature (121°C) under pressure ( 15 lbs/in2)

Lecture 3 – Use, Growth and Enumeration of microorganisms in Molecular biology •

Microorganisms used as tools in molecular biology

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for studying genes, you would extract the cell, create as a microorganism and then examine its function

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Mutations in genes can be created to see how it effects protein structure – used for gene therapy

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Saccharomyces are a type of yeast.

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The type of organism that you are using is dictated by the experiment and by what you want to do and accomplish in the lab

Advantages of these Microorganisms •

Biology/genetics well studied

– well-defined genetic system – mutant isolation relatively straight forward (number of mutants available) •

Genome sequencing complete

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Easy to grow/rapid (grow rapidly in lab settings) – dispersed cells (single celled) – grows in liquid and solid media – can grow in bulk – form discreet colonies on solid media

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Easy to manipulate

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Can store frozen stock

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A highly versatile DNA transformation system – can be used to store via transformation foreign DNA – can be used to express foreign gene product

Escherichia Coli (E. coli) •

Gram-negative, rod-shaped bacterium (~2 mm in length including flagella) with a genome of 4,639,221 base pairs encoding at least 4000 genes

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The K12 (wild) strain was first isolated in 1921 from the stool of a malaria patient and it has been maintained as a pure strain in laboratory stocks for the last 100 years. – molecular biology strains are derived from K12 via mutations •

virulence factors removed and built in mutations that prevent growth outside of the lab

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E. coli laboratory strains can support the replication of DNA plasmids, many of which encode antibiotic-resistance genes

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Many laboratory strains (>100) are commercially available

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Restriction modification systems have been removed from E. coli K-12 strains because they will interfere with the replication of foreign DNA in bacterial cells – In wild type E.coli the bacteria will degrade foreign DNA as a defense mechanism! – Therefore, the strains used in molecular biology are mutated

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Genetic variants of E. coli K-12 have led to improved strains for recombinant DNA methods: 1.Bacterial restriction modification systems have been removed. 2.DNA recombination systems are modified to prevent rearrangements. 3.Endonuclease activity has been mutated to increase plasmid yields – endonuclease are enzymes that will degrade DNA – the are made to not digest foreign DNA

Saccharomyces cerevisiae •

One of the most ideal eukaryotic microorganism for research. Typically, approximately 810 mm diameter spheroids/ellipsoid – Relative to E. coli these are very large

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Greater genetic complexity than bacteria, containing 3.5 times more DNA than Escherichia coli cells (16 chromosomes in haploid, 12.1 million bp, ~6300 genes)

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A type of nonpathogenic yeast - can be handled with little precautions – Non-pathogenic mean it wont make you sick.

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Unlike most other microorganisms, strains of S. cerevisiae have both a stable haploid and diploid state (can use for genetic mating experiments )

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Many types of well characterized strains can be obtained commercially (e.g. temperaturesensitive, nutrient sensitive (auxotrophic)

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Normal laboratory haploid strains have a doubling time of approximately 90 min. in complete YPD (1% yeast extract, 2% peptone, and 2% glucose) medium and approximately 140 min in synthetic media during the exponential phase of growth at the optimum temperature of 30°C.

Growth •

increase in cellular constituents

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may result in increased organism size, population or both

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bacteria usually reproduce by binary fission •

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binary fission- cells enlarge and divide to yield two progeny of approximately equal size

S. cerevisiae grows by budding (asexual), daughter cell buds from mother cell

Budding vs Binary fission

Factors Affecting Growth: 1. Chemical and physical nature of their surroundings - controlling these parameters can ensure optimized growth rate of any microorganism 2. Genotype: different genus/species/strain can grow at different rates - E.coli- doubling time is approx. 20 min at 37-39C/optimal nutrients - S. cerevisiae - Doubling time of approximately 90 min. at 30°C 3. Chemical factors in the ...