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