Biology Lab Final

sorry this is not going to be finished i already took this exam but uhhhhh if you want me to finish it i will

micropipette

tool to obtain precise volumes; NEVER turn the indicator dial beyond the upper or lower volume limits of the micropipette

P1000

measures volumes between 100-1000 μl; 1 μl adjustment

blue

P100

measures volumes between 10-100 μl, 0.1 μl adjustment

yellow

P10

measures volumes between 0.5-10 μl; 0.01 μl adjustment

red

microliter (μl)

most used unit for measuring and dispensing volumes in molecular biology; 1 μl = 10^-6 L = 10^-3 mL

micropipette anatomy

• plunger - topmost part of the micropipette which is used to expel and take up the desired amount of liquid into micropipette tip

• eject button - used to eject the tip from the micropipette without touching the micropipette tip.

• volume display window - adjusted volume is shown

• tip attachment - where tips attach

Pipette tips

tips attached to the tip attachment for taking in liquid and then transferring it from one place to another without contaminating the micropipette

first stop (or soft stop)

used to fill the micropipette tip (or release in a gel)

second stop (or hard stop)

used to dispense the contents of the tip (except in a gel)

Accuracy

micropipette delivering the correct volume; giving results near to what is true or expected

Precision

produces reproducible results; low standard deviation

BEST PRACTICES WHEN PIPETTING

1. Do not withdraw the liquid too quickly into the pipet.

2. Slowly allow the plunger to return with your thumb.

3. When collecting your solution, DO NOT push past the “first stop”

4. Depressing the plunger to the second stop before filling the micropipette tip.

5. Release plunger slowly

6. Dispense liquids against the side of the tube or directly into the liquid in order to eject the full volume

7. Don’t hold the pipette when you’re not pipetting

Bradford assay

colorimetric method used to determine the protein content in a solution based on the formation of a complex between the dye, Coomassie blue, and proteins in solution

Coomassie blue

As proteins bind the dye, there is a change color of from brown to blue; recorded using a spectrophotometer at a wavelength of 595nm

Beer-Lambert law

absorption is proportional to the amount of dye bound and hence the amount of protein in the solution

T = IT/I0

A = -log T

A = ϵbc

spectrophotometer

separates white light into a spectrum of colors (wavelengths) and measures the amount of light absorbed and transmitted by a dissolved chemical; performs Absorbance, % Transmittance, and Concentration measurement

cuvette

contains the sample to be measured; arrow on the front of the cuvette should be oriented in the direction of the light path

Dilution

process of decreasing the concentration of a solute in a solution; mixing it with more solvent like adding more water

direct dilution

adding solvent once to get desired concentration; split up DF into multiple factors of dilution

serial dilution

multiple additions of solvent to get to desired concentration

dilution factor (DF)

Final concentration / Initial concentration

standard curve

plot of absorbance vs. a varying amount of some known concentration of proteins

scientific method

framework used to accurately investigate the world and explain natural phenomena

hypothesis

proposed explanation for a phenomenon based on observations and existing knowledge; leads to experimental predictions

Predictions

often written as "If X then Y" statements

Experiment

procedure designed to test a hypothesis; must be reproducible and reliable, include a control, and use well-designed variables

independent variables

changed by the researcher; 1 per experiment

dependent variables

affected by the independent variable and that is measured; no limit to how many

controlled variables

held constant during the experiment

controls

will not receive the treatment (neutral condition or receive placebo)

experimental group (Test group)

receive the treatment/change in independent variable

Positive controls

groups where the condition guarantees a positive result

Negative controls

produce a negative outcome

Reproducibility

Anyone should be able to replicate the experiment and get similar result

Model organisms

species that are used to study certain aspects of biology

• Easy to breed and maintain in a laboratory setup

• Short generation times

• Large number of offspring

• Well studied genome

  • Can be manipulated easily

• Decreased complexity compared to humans

• Efficient manipulation of genomes

• Less ethical concerns compared to research on human

levels of biological organization

Biosphere > ecosystem > community > population > organisms > Organs > tissues > cell > organelles > molecule

enzyme

macromolecule that acts as a catalyst; affected by temperature and pH

catecholase (or catechol oxidase)

common in plants; fruit browning reaction; forms benzoquinone and water from catechol; stored in vesicles in undamaged cells

transgenesis or genetic transformation

universality of the genetic code allows genes from one species to be transplanted into the genetic code of another

transformation

1. host into which DNA can be inserted (E. coli)

2. Vector (plasmid)

3. Selection method or selecting and isolating the successfully transformed organisms (ampicilin/blacklight)

Vector

means of carrying the DNA into the host

• Origin of replication (ori) - allows the plasmid to be recognized by the replicating machinery of the bacteria. This will allow the plasmid to replicate in the host.

• Restriction enzyme sites – allows ligation of the DNA fragment (gene of interest) into the

  plasmid.

• Antibiotic resistance - allows the identification of a positive transformant

competence

natural ability to alter their genetics by taking up extracellular ("naked") DNA from their environment; promoted by heat shock or electroporation

CaCl2 (calcium chloride) in transformation

eutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane is used to allow the DNA to enter the cell; increases cellular competencyhe

heatshock

chemically competent cells are mixed with plasmid DNA in ice, briefly (25-45 sec) exposed to an elevated temperature (usually 42oC), then immediately returned to ice (0°C) for ≥2 minute. The rapid temperature change creates a thermal imbalance on either side of the cell membrane, creating pores that moves the plasmid into a small percentage of cells

Aseptic technique

designed to provide a barrier between the microorganisms in the environment and the sterile cell culture

• Before and after use, your work surface should be disinfected thoroughly.

• Always keep the lid on your plates, except when performing manipulations.

• When transferring bacteria, sterilize the loop or you are use a sterile disposable loop.

• Avoid touching the pipette tip to anything non-sterile, including the outside edge of the bottle

colony

visible mass of cells usually resulting from the division of a single cell and the number of cells in a single colony can exceed one billion (10⁹); growing very close to each other fuse into lawn

promoter

where RNA polymerase binds and begins transcription of the gene

inducible promoter

can be switched from an OFF to an ON state

operons

clusters of genes controlled by a single promoter

Gene green fluorescent protein (GFP)

reporter gene from jellyfish

central dogma

DNA➜RNA➜PROTEIN➜TRAIT

phenylthiocarbamide (PTC)

bitter tasting chemical similar to those found in vegetables

PTC taster allele

dominant

PAV (ProAlaVal)

cut by restriction enzymes

PTC nontaster allele

recessive

AVI (AlaValIle)

not cut by restriction enzymes

Genomic or chromosomal DNA (gDNA)

full-length DNA sequence, including coding (exons) and non-coding (introns) DNA; majority of DNA in a cell is found in the nucleus, with a much smaller amount in the chloroplasts and mitochondria; typically more mitochondria per cell than nuclei per cell so one finds more molar equivalents of DNA in the mitochondria than in the nucleus; can be harvested from any sample containing eukaryotic cells apart from a pure sample of mature red blood cells

Mitochondrial DNA (mDNA)

found inside the mitochondria of cells and differs from the nuclear DNA in that it comes from the mother only

Complementary DNA (cDNA)

not found inside cells but is created by reverse transcription of RNA in a test tube

centrifugation

technique that helps to separate mixtures by applying centrifugal force; substances separate according to their density; ALWAYS BALANCE THE CENTRIFUGE with hinges out

pellet

solid particles at the bottom of centrifuge tube

supernatant

remaining solution in centrifuge tube

Lysis Buffer

• Detergent (such as SDS or Sodium Dodecyl Sulfate) will lyse the cells by solubilizing (breaks apart) the phospholipid cell membrane and nuclear membranes, allowing the DNA to be released

• buffering agent (Tris) to maintain the pH of the solution so that the DNA stays stable

• Protease (usually proteinase K), an enzyme that digests proteins removes proteins bound to the DNA (such as histones) and destroys cellular enzymes that would digest the DNA

  • incubated at 50 -55°C, the optimum temperature for protease activity

Salt solution (NaCl)

added to help precipitate proteins and cellular debris clump together

Alcohol (isopropyl/ethanol)

isolate concentrated DNA (Precipitation); esp. cold

TE (Tris -EDTA) buffer

• TRIS, pH 8.0 (hydroxymethyl) aminomethane is a buffering agent used to maintain pH.

  • EDTA (Ethylene Diamino Tetra-acetic Acid) chelates any divalent cations like Ca2+ or Mg2+ preventing the activity of DNase (nucleases which require these ions to be active)

POLYMERASE CHAIN REACTION (PCR)

used to replicate DNA of a specified length in vitro

• Primers (forward (5' to 3') and reverse (3’ to 5’))

• Nucleotides

• DNA polymerase

• DNA template

• Extra care with contaminations

• A thermocycler

Primers

bind at a specific DNA sequence and mark the beginning of the DNA amplification

Taq polymerase

active at temperatures up to 95oC; multiple cycles of PCR can be performed in a single continuous event, and no additional polymerase is required

PCR Reaction

• Denaturation (melting the double stranded DNA into two separate strands) >94oC

• Annealing (primer binding) at 50oC to 65oC

• Elongation. (DNA synthesis) at 72oC

copies of replicated DNA

2^n - 2n

Restriction Fragment Length Polymorphism (RFLP) analysis

enzyme digestion followed by electrophoresis

gel electrophoresis

process that separates molecules using an electric current

• Semi-solid, porous gel matrix

• DNA or RNA sample

• Loading buffer

• Dye

• Molecular weight standard samples or "ladders”

TASTE TEST

phenotype testing

evolution

Change in allele frequency over time

Hardy-Weinberg equilibrium

• No gene mutations may occur and therefore allele changes do not occur.

• There must be no migration of individuals either into or out of the population.

• Random mating must occur, meaning individuals mate by chance.

• No genetic drift, a chance change in allele frequency, may occur.

• No natural selection, a change in allele frequency due to environment, may occur

Caenorhabditis elegans (C. elegans)

a small, free-living, nematode worm (round worm) that is currently used as a genetic model; feeds on microbes such as bacteria; Most of these nematodes are self-fertilizing hermaphrodite and a few are male

C. elegans development

4 days; Juvenile worms progress through four larval stages (L1-L4) over the next two days, increasing in size with each stage. After the fourth larval molt (L4), the worms are reproductively mature, meaning that they can be used for further genetic studies. Adults will live for 2-3 weeks, over which time they gradually age and lose vigor

C. elegans as model organism

a simple and complete genome, a fast generation time, and are easy and inexpensive to maintain; 35% of worm genes have human homologs; extensive nervous system

SEXING of C. Elegans

• Hermaphrodite - sharp tail, visible embryos in center

• Male - spade tail, no eggs

mating of C. elegans

s. Males arise by spontaneous X chro mosome nondisjunctionat a frequency of ∼0.1%

C. elegans mutations

• Him-5 - increased rate of X-chromosome nondisjunction, leading to a higher proportion of male progeny (Up to 30% vs. 0.1% wildtype incidence)

• Rol (rolling) - rolls around in crcles aimlessly

• hbl (hunchback) - bent on dorsal side

• Dpy (dumpy) - short and stout

• bli (blister) - balloon like blisters on head and body

• adr-2 - no ADAR activity and have lower protein diversity so aplastic and unadpative

Aicardi-Goutieres syndrome

mutations in the ADAR gene in humans; early onset

childhood disease characterized by microcephaly, seizures, glaucoma, skin lesions, and general abnormal

neurology

Exposures to heavy metal

common and dangerous environmental health issue.

The fast metabolic rates and limited self-repair abilities of nerve cells, as well as the large number of chemical messengers used in interneuron communication, make the nervous system particularly vulnerable to these chemicals