Bio lab exam Rutgers

Assay

tool used to quantitatively measure the amount of a single part of a total sample (glucometer to measure glucose)

Accuracy

how close a measurement is to its true value

(how close are you to the bulls eye?)

Precision

ability to repeatedly measure a value in a fixed situation and get the same results

(how many times did you get close to the bulls eye?)

Significant figures

digits that carry meaning contributing to its measurement resolution

Proteins

dependent on pH, salt concentration, and temperature of the reaction mixture (10 degree change equals 1000 decrease in change)

Buffer

an aqueous solution containing a specific mixture of salts, buffering agents, reducing agents, and cofactors

resist changes in concentration of H ions

Stock solutions

concentrated solutions that last over long periods of time: take up less space: easily diluted for use with water

C1V1=C2V2

to make dilute solution

Dilution factor

how the [ ] of the dilute solution is reduced compared to the [ ] of the stock solution

DF = C1/C2 = V2/V1

Spectrophotometer

spectrovis

a machine that measures absorbance/transmittance of a pigmented solution, commonly used to quantify the [ ] of materials in a solution

the absorbance of a sample is directly proportional to the concentration of material in a sample

use of a blank to calibrate spectrovis (zero absorbance)

Graphs

must have title, independent variable on x axis, dependent variable on y axis, labels on axes, a legend

Discovery science

uses large amounts of data or surveys of natural systems to discover patterns and correlations

Science

"to know"

Standard solutions

have a known amount of material and are used to calculate amount of material in the unknowns

1) Cs/Cu = As/Au

2) Standard curve: find [ ] of unknown by using trendline:

linear regression: to model relationship between x and y

R^2: how well trendline fits the data (0.97 or better is good)

Hypothesis driven science

scientific method

problem/observation, background information, hypothesis, prediction (if-then), test predictions, conclusions, report conclusions

hypotheses can NEVER be truly right

Independent variable

what is changed by researcher

Dependent variable

respond to changes and is measured

Controlled variables

factors that must be controlled because they could affect the dependent variable

Experimental group

independent variable added here

Qualitative data

not numerical

Quantitative data

numerical, use statistics (identify before experiment happens) i.e. frequency of distributions, measures of central tendency, analysis of variance

Enzymes

organic catalysts that lower activation energy for a reaction to take place

**optimal temperature and pH, end in -ase

1) binds to a substrate: substance the enzyme acts on

2) substrate binds to active site: enzyme-substrate complex

3) enzyme changes the substrate to make the product: substance that forms because of the enzyme catalyzed reaction

Temperature

the average kinetic E of particles regardless of volume

pH

-log[H+]

acidic: H+ > OH- closer to 0

base: OH- > H+ closer to 14

cytochrome c oxidase enzyme

oxidase that makes reduced cytochrome c (pink) go to oxidized cytochrome c (red)

required for mitochondrial respiration

cytochrome c oxidase

in complex IV of the ETC in the mitochondrion/ plasma membrane

required for mitochondrial respiration

highly conserved

from liver of beaver

Rate

change in quantity per unit time

Reaction rate

|changeA| / changeT

Biuret method

to evaluate the concentration of protein present (complex turns blue to purple)

Population

the entire group of organisms, used to characterize variability in a system, but broken down into a sample

Subset

subset of population for observation

Descriptive statistics

describe and summarize data

1) measure of central tendency (mean, median, mode) to test central position in data

2) measures of dispersion: (range, variance, standard deviation): describe variability of values

Distributions

describe the frequency of each value in the sample

normal distribution = bell shaped curve

Inferential statistics

to make estimates and draw conclusions (sample vs larger population)

T test: p value:

1) x > 0.05: not significant

2) x < 0.05: significant

Water quality index (WQI)

1970 by National Sanitation Foundation- quantify and track quality of water sources

9 Parameters: temperature, pH, turbidity, total solids, phosphates, dissolved oxygen, biochemical oxygen demand, fecal coliform

Q value

varies 0 to 100 from the graph, multiplied by weighting factors

Weighted Q values added together to get water quality index score

Temperature, pH, BOD weighting factors

0.11

Total phosphate, nitrate weighting factors

0.10

DO weighting factors

0.17

Fecal coliform weighting factors

0.16

Turbidity weighting factor

0.08

Total solids weighting factors

0.07

WQI is better for _________ waters

moving waters (lotic)

Lotic source

Stream/river

Lentic source

Pond or lake

Temperature WQI

more gas can be dissolved in cold water than in warm water

increased temp = increased photosynthetic rate = increased plant growth and algal blooms = bad

pollution: industrial water returns warmer, removing shade trees, T of or above water

water cooled by cold air temperatures, introduction of colder water

too high/cold= stress= lowered resistance to pollutants, diseases, parasites

pH WQI

organisms sensitive to, if too low/high may not survive or reproduce

changes in pH from algal blooms (more basic), industry (raise/lower pH), rainfall on acidic minerals (more acidic)

Total solids WQI

all the suspended, colloidal, and dissolved solids (including dissolved salts like NaCl and silt/plankton)

siltation is the most common pollutant of streams and rivers

soil erosion, runoff (fertilizers, water treatment, parking lots), catfish (stir up bottom sediment), OM

=high: reduce clarity (decrease sunlight, increase temp)

too many salts= dehydrated organisms

Dissolved oxygen WQI

available to aquatic organisms, measured in % saturation or mg/L

affected by temperature, stream flow, air pressure, aquatic plants, decaying OM and human activities

change during day because of photosynthesis, highest in the afternoon and lowest at night

must be tested at the same time

Large fluctuations in short time = algal bloom

[high]: trout, salmon, mayflies

[low]: catfish, mosquito larvae, carp

BOD WQI

the decrease in dissolved oxygen due to oxygen being depleted faster than it can be replaced

1-2 mg/L= clean

x>10mg/L=very poor

dependent on OM: death of aquatic organisms, rain transporting OM from soil to water, leaves, animal waste

IF OM decompo is too high, DO can be severely reduced (bad)

Morphospecies identification

use of morphological characters (structure, form, shape, color, length) to identify the organism

DNA barcoding

use a standard molecular marker/short sequence of DNA to get organisms species

Plankton

suspended in the water column (microscopic plants and animals)

phytoplankton: primary producers

zooplankton: primary consumers

Primary source

peer reviewed, scientific journal article

Primary literature

comments on the immediate results of research activities (theses, technical reports)

Nitrates WQI

freshwater: x<1mg/L

pollution with nitrates: animal feed, ag runoff, treatment water

Eutrophication: high nitrate concentrations leads to increased algal blooms/cyanobacteria

Blue baby syndrome (fatal-infants): x>10mg/L

Phosphorous WQI

limiting factor in plant and algal growth

Excess leads to eutrophication: lowers levels of DO to uninhabitable levels, increases BOD

1) orthophosphates: inorganic, fertilizers

2) organically bound: decaying OM, animal wastes

3) condensed: detergents, water supplies to prevent corrosion

4) total: all (most commonly reported)

Fecal coliform (WQI)

to see if E coli is in the water (bacteria that is in intestinal tracts of animals and humans)

E coli as indicator of pathogens of fecal origin = human health risk

High E coli: leaking septic/sewer, wading cows/waterfowl, polluted runoff: good sewage indicator because not usually in water or soil, easy to ID, lives a little longer in water than other bacteria

Presumptive test

infer amount of gas produced by E coli present:

--assumes it's even being produced by E coli

could be other coliform bacteria (facultative anaerobes that ferment lactose to produce gas)

Collected in small Durham tubes

incubated at 44.5 degrees because other coliform can't survive the temperature

Total phosphate WQI

1) digest sample

2) spectrophotometer: to create standard curve

Look at genome sequence to determine the biological species, sequencing genome expensive

Conserved region of DNA

nucleotide sequence that has little to no variation across species- remained relatively constant throughout evolution

DNA barcoding

purify genomic DNA from samples, amplify with PCR using DNA primers, Agarose gel electrophoresis to see if PCR is successful, Choose samples to DNA sequence

Search databases to find out organism

Agar

solid matrix of agarose and agaropectin with nutrients (grow medium)

bacterial samples

no lysis buffer- the heat in PCR will lyse the cells

analyze euk dna

extract(break down tissue in bead tube homogenizer), chemical/enzymatic digestion follows

PCR amplification

small amount of template DNA

Denaturing: 94 degrees to release DNA

purify DNA to prevent denatured proteins/lipids stopping reaction

DNA column based extraction

DNA binds to column due to ionic forces

Wash buffer (Ethanol) to remove any contaminants (RNA, salts, lipids)

Water added: water contains the isolated genomic DNA

Sterile technique

procedures to prevent contamination of the solutions and cultures

avoid prolonged exposure to the air

cover pipet boxes

don't use cloudy media

Pick single colony of bacteria and put into a luna broth (liquid media) to grow

Turbidity WQI

water's lack of clarity

--high= cloudy

--low= clear

Bottom dwelling organisms (catfish), suspended OM, increase in stream flow making increase in soil erosion, runoff (industrial and ag)

High turbidity decreases amount of sunlight that penetrates water --> decreases photosynthetic rate, increases temperature

Less aesthetically pleasing

PCR

1) DNA polymerase: synthesize new DNA

2) dNTPs: for incorporation

3) short oligonucleotides (primers) that DNA polymerase adds the dNTPs to extend the DNA strand

4) DNA for a template

Taq polymerase

thermophilic bacteria that won't degrade at 95 degrees; Thermus aquaticus

Denaturation

95 degrees: purified genomic DNA put in microcentrifuge tube: disrupts H bonds holding strands together

Primer annealing

55 degrees

Two primers

1) P1: homologous to gene at 5' end

2) P2: homologous to gene at 3' end

Synthesis

72 degrees

Taq synthesizes DNA from two primers (3' ends of both point towards the gene): the DNA between the primers is replicated

2^n, where n=number of cycles

Primers

15-20 nucleotides long

Forward and Reverse hybridize on ends of regions we are amplifying: only amplify DNA in between the primers

--won't anneal to other sequences: only the conserved regions

-- anneal with the opposite strands of the denatured gene

DNA barcode marker: COI gene

cytochrome c oxidase

mitochondria and respiration

700 bp fragment (animal or plant)

DNA barcode marker: rbcl

ribulose-biphosphate carboxylase

chloroplast genome = carbon fixation

in photosynthesis, plant specific

600 bp

DNA barcode marker: 16S

16S ribosomal RNA: bacteria only

1500 bp

does not need to be purified for PCR

Master mix for PCR

DNA polymerase, dNTPs, buffer, forward and reverse primers, water

Reduce pipetting errors and time spent running the reaction

Three different mixes (one for each primer set)

Aliquot

small portion of genomic DNA or bacterial culture that is added to the PCR tube

2X Taq mix

enzyme (Taq), buffer, and dNTPs

--Add nuclease free water, primers, and DNA

CFU Assay

serial dilution of water sample in microfuge tubes --> small volume onto LB agar plant

Incubated bottom side up to prevent overgrowth

CC= cannot count

Loading dye

glycerol and pigments: xylene cyanol, bromophenol blue

Glycerol denser than water: DNA sample sinks to bottom of well

monitors how far the DNA migrated

EtBr

ethidium bromide

added to visualize DNA fragments -- distorts when it binds (carcinogen)

migration of DNA

black (-) ----migration of DNA-----> red (+)

gel electrophoresis

to see if PCR was successful and size of the resulting DNA fragments

-Separate, identify, and purify DNA fragments

-net negative charge: if DNA is put into an electric field, DNA molecules will migrate towards the positive electrode

Fragments are forced to move through agarose: smaller fragments move faster and further

Analysis of the gel as a whole

1) only ladder and no sample bands: Taq/dNTPs degraded in master mix

2) no bands and no ladder: EtBr not added to the gel

3) no bands for a sample: a primer not added to the master mix

Gel as a whole: only ladder and no sample bands

Taq/dNTPs degraded in master mix

Gel as a whole: no bands and no ladder

EtBr not added to the gel

Gel as a whole: no bands for a sample

a primer not added to the master mix

Analyze Gel as a Quadrant: no bands (animal/plant)

DNA extraction recovered little or no genomic DNA/ genomic DNA not added to PCR

Analyze Gel as a Quadrant: no bands (bacteria)

bacterial culture not properly inoculated/ bac culture not added to PCR

Analyze Gel as a Quadrant: more than one band in one lane

1) non specific primer binding: primers hybridize and initiate synthesis (? possibly from contamination)

2) COI added to plant/bacteria

Primer dimer

primer molecules that hybridized to each other

-visible after gel electrophoresis

-30-50bp

Evolution

the process of change that has transformed life on Earth- fundamental organizing principle of biology

Biology

scientific study of life

Levels

biosphere -> ecosystem -> communities -> populations -> organisms -> organs + systems -> tissues -> cell -> organelles -> molecules -> atoms

Emergent properties

absent from the preceding level, due to the arrangement and interactions of parts and interactions of parts as complexity increases (chlorophyll a and b --> chloroplast)

Systems biology

the exploration of a biological system by analyzing the interactions among its parts

Gene expression

information in a gene directs the making of a cellular product

Transcription ---mRNA----> Translation ---ProteinFolding--> Protein