CTE Skills Test Review
LAB ATTIRE
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Closed toe shoes
Lab coat
Goggles
Gloves
Hair tied back
No cosmetics
No gum, food, or drink
DISPOSAL
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Glass
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Bacteria
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Glass waste disposal box
Bleach
Autoclave
Other waste
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Proper garbage disposal
LAB NOTEBOOK
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Date
Initials
Number each page
Table of contents
LABELING
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Reagent bottles, bacterial plates, etc.
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Initials
Date
What is in and/or on
Concentration (M or %)
On the bottom of the plate [part with agar]
ASEPTIC TECHNIQUE
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Disinfect counters and work surfaces.
Flaming loop, spreader, etc.
DISINFECTION
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Always clean counter before and after
Use
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70% ethanol
Bleach
Other disinfectants
EFFECT OF UV ON BACTERIA
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Kills bacteria if you leave it under UV light
AUTOCLAVE
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Uses high heat and pressure to sterilize
objects
MICROPIPETTES
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1000 ul = 1 ml
Be within the range but not on the end with
amounts to pipette
P20, p100, p200, p1000
CHEMISTRY EQUATIONS
 Molarity
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moles = grams/molar mass
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Molar mass is the sum of the atomic mass of
elements
M
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(M) = moles/liter
= [grams/molar mass]/liter
Volume % = volume solute/volume solution x
100
 M1V1
= M2V2
 C1V1 = C2V2
BONDS
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Hydrogen- hydrogen bond with other element
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Covalent- sharing of a pair of electrons by two
atoms
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ions
Buffers- salt, etc.
Disulfide- binds peptide chains
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DNA on backbone
Ionic- through transfer of 1+ electrons
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Water, DNA between nitrogen bases, proteins
Gives protein 3D shapes [usually between chains]
Peptide- bond amino acids, remove a molecule of
water (dehydration)
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Amino acid bonds
HYDROPHOPIC/HYDROPHILIC
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Part of the membrane
Hydrophobic
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Water fearing
Tails
Hydrophilic
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Water loving
Head
POLAR/NON-POLAR
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Polar
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Has a charge
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+/Ex: water
Non-polar
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Doesn’t have a charge
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Ex: sugars, oils
pH
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Salt concentration can change the shape of
proteins
Change in acid and bases can kill enzymes
Acids
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Lower pH to 1>7
Bases
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Raise pH to 7<14
STOCK SOLUTIONS
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Dilute stock solution to get desired solution
concentration
C 1V 1 = C 2V 2
SPECTROPHOTOMETRY
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An instrument used to determine the intensity
of various wavelengths in a spectrum of light
Can determine concentrations of solutions
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Can make a graph of OD and absorbance v.
concentration
Can change the wavelength to find the protein,
DNA, RNA, and bacterial concentration
SERIAL DILUTION
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Dilute to get smaller and smaller concentrations
Can go from lawns to single colonies, an alternative
to streaking
PROKARYOTIC V. EUKARYOTIC
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Prokaryotic cells
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No nucleus
Eubacteria
Archaebacteria- extreme
bacteria
Operon (grouped genes)
Don’t have introns
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Eukaryotic cells
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Nucleus
Protists
Fungi
Plants
Animals
DNA has introns and
exons (splice introns out
and bind exons together
to form mRNA)
MEDIA PREPARATION &
INCUBATION
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Media preparation- can manipulate what you grow
Incubation
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37°C if grown inside body
25°C if grown elsewhere
LB/AMP/ARA
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LB- nutrients necessary for bacterial growth
AMP- ampicillin resistance to see if there was an uptake of
the protein
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Selective procedure- only ampicillin resistant bacteria can
grow
ARA- arabinose to turn on the GFP
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Allows the gene to be transcribed (operon involved)
ANTIBIOTIC RESISTANCE FOR
SELECTION
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Antibiotic- natural substance secreted by 1
microorganism that will kill or inhibit growth
and reproduction in other microorganisms
Shows if there was an uptake of the new
genes
INOCULATION OF MEDIA
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Streaking
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Proper lab attire…not that kind of streaking!
Get a single colony on you loop and streak it
across the plate in a zig-zag fashion. Turn it a
quarter turn, flame and repeat.
Spreading
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Pipette LB broth onto plate and sterilize paperclip.
Spread broth with paperclip over the entire gel.
IDENTIFYING MICROORGANISMS
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Colony Morphology
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Size
Shape
Margin
Elevation
Texture
Light transmission
Color
Antibiotic resistance
Incubation temperature
Gram staining
BACTERIA TYPES
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Cocci
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Bacilli
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Grape-like clusters
Strep
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rod
Spiral
Staph
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round
Chains
Diplo
Two together
GRAM STAINING
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Depends on the structure of
the cell wall
Gram positive
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Purple
Gram negative
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Red
DNA STRUCTURE
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Runs 5’ to 3’
Sugar (deoxyribose)
Phosphate (phosphoric acid)
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Negative charge (allows for electrophoresis)
Nitrogen bases
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2 hydrogen bonds
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Adenine – Thymine
Gene cutting happens most often here
3 hydrogen bonds
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Guanine – Cytosine
DNA REPLICATION ENZYMES
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Helicase
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RNA primase
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Splits the DNA molecules apart
binds primers (RNA nucleotides) by complimentary base
pairing
DNA polymerase
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Adds nucleotides to extend the DNA
Binds leading DNA strand starting at 3’ end
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TAQ polymerase can withstand high heat
RNA primers are removed
Ligase
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Seals gaps in the sugar phosphate backbone
RNA
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Ribose sugar
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has one more oxygen molecule
Phosphate
Nitrogen bases
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Adenine – URACIL
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not thymine
Guanine – Cytosine
TRANSCRIPTION
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DNA to RNA
Eukaryotes
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Eukaryotes have introns
and exons; the introns
are removed
5’ cap and 3’ poly-A tail
on the exons that have
been spliced together
Prokaryotes
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Operons
PROTEINS
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Peptide bonds
Eukaryotic
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Multiple proteins from 1 RNA
Prokaryotic
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Operon
PROTEIN STRUCTURE
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Primary
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Secondary
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Alpha-helices or beta-sheets
Tertiary
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Amino acid sequences
Domains
Quaternary
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Subunits
FUNCTIONS OF PROTEINS- REST!
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Regulatory
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Enzymes
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All enzymes are proteins but not all proteins are enzymes
Covalent bond breakage and formation
Structure
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Genes and cellular processes are turned on and off
Mechanical support to cells and tissues
Transport
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Move things in and out of the cell
PROTEIN SYNTHESIS
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RNA to protein
Initiation
Elongation
Termination
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mRNA
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coded DNA
Codons
tRNA
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The stop codon doesn’t
code for an amino acid
transfers amino acid
Anti-codons
Amino acids
AMINO ACID CHARACTERISTICS
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Peptide bonds (dehydration)
Water
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Charge
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Hydrophilic
Hydrophobic
Positive
Negative
Polarity
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Polar
Non-polar
Uncharged polar
DNA FINGERPRINTING
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Identifies matching
DNA fragments (bands)
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RLFP- Restriction length
fragment polymorphisms
VNTR- Variable
Nucleotide tandem
repeats
Introns- non-coding
sequence that varies
RESTRICTION ENZYMES
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Also called endonucleases
Specific sequences of DNA nucleotides
Cut at specific places
Can be palindromes (same forwards and
backwards)
Come from bacteria
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To cut up viral DNA
Methylate own DNA to protect it
RESTRICTION DIGEST
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Where you cut DNA with restriction enzymes
Results in DNA fragments
Can then be run on gel electrophoresis
GEL ELECTROPHORESIS
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Gel electrophoresis- uses electric current to
separate DNA fragments by size
Runs to red (positive)
Bands
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Various sized fragments of DNA
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Introns
Pieces
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Smallest ones run the farthest
SDS-PAGE ANALYSIS
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Run vertically
Mostly used for protein
Smaller pores than agarose
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Smaller matrix
Sorts by size and charge
PCR
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Polymerase Chain Reaction
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Denature
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Anneal
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Raise the temperature to unzip DNA
attach primers
Extend
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Binds TAQ polymerase
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TAQ polymerase can withstand high heat
Heat and cool
1 million copies for 30 cycles
DNA SEQUENCING
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Used to know the nucleotide sequence of the
human genome
Process
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Put DNA with DNTPs
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Terminates elongation of DNA
PCR
Run on a gel to tell the sequence of the
nucleotides
READING FRAME
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Frame shift mutations
Point mutations
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Deletion
Insertion
RECOMBINANT DNA
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2 different pieces of DNA combined
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Use restriction enzymes to cut the gene of interest
and the plasmid
Insert the gene of interest into the plasmid
TRANSFORMATION
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Insertion of a gene into bacteria
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Competent cells- take up the plasmid
Restriction enzymes- cut the plasmid
Selection- so you can get the colonies with the
selected gene
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Antibiotic resistance
PLASMID
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Origin of replication
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Antibiotic resistance
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Allows for the selection of the desired bacteria
Operon
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Allows plasmid to self-replicate
Turns on the gene of interest
Gene of interest
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Example: GFP