Introduction to Cell and Molecular Biology Laboratory Flashcards
The total length of SARS-CoV-2 S is _________ and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the S1 subunit (14-685 residues), and the S2 subunit (686-1273 residues)
1273 aa
_________ visually form a characteristic bulbous, crown-like halo surrounding the viral particle
S protein trimers
are an important class of secondary metabolites known for their protection agaisnt UV radiation and other stress factors
Mycrosporine-like amino acids (MAAs)
Cyanobacteria produce a variety of MAAs, including ________, the active ingredient in many sunscreen creams
Shinorine
Topics under cell and molecular biology
- Molecular mechanisms inside the cell.
- Flow of information within and between cells.
- Intracellular and transmembrane transport.
- Control of gene expressions.
- Survival and death of cells.
- Formation of cancerous cells.
Course Learning Objectives
After finishing this course, you should be able to have:
- In-depth understanding of the basic and advanced concepts in cell and molecular biology.
- Learn and apply the recent research techniques/approaches in the field
- Develop research skills and techniques for critically evaluating scientific literature.
Routine Workflow in a Molecular Biology Lab
- Collecting samples, preparing samples
- Extracting DNA/RNA/proteins
- Determining DNA/RNA/protein concentration
- Amplifying gene of interest (GOI) by Polymerase Chain Reaction (PCR)
- Detecting DNA/RNA/protein by gel electrophoresis
- Documenting gel images (“Gel Doc”)
- Downstream analysis: DNA sequencing or cloning, etc.
DNA Workflow (arrows)
- DNA Extraction
- Pure DNA
- (AGE/Spectrophotometry = DNA Check: Purity, Quality, Quantity)/PCR
- From PCR -> AGE/Spectrophotometry
- From AGE -> PCR product purification
- Sequencing, cloning, other applications
DNA Analysis Workflow
DNA Fingerprinting/DNA Barcoding
- Multiple Sequence Alignment ->
- Phylogenetic Tree
RNA Analysis Workflow
Gene Expression Profiling
- Cell culture
- Cell lysis/RNA Isolation
- Reverse transcription
- Quantitative PCR (qPCR)
a technique that amplifies and simultaneously detects the amplification product of nucleic acids, allowing for the quantification of DNA or RNA
Quantitative PCR (qPCR)
The Molecular Cloning Workflow
Vector Preparation -> Insert Preparation -> Ligation -> Transformation -> Colony Screening
Gene from a plasmid/DNA fragment is ligated into a vector (another plasmid) = Recombinant DNA technology
bacteria carrying both the WT gene and the integrated plasmid
merodiploid state
Molecular cloning workflow wherein this happens:
- Restriction digestion
- Dephosphorylation
- Blunt-end creation (optional)
- Purification
Vector Preparation
Molecular cloning workflow wherein this happens:
- Restriction Digestion
- Blunt-end creation (optional)
- Purification
Insert Preparation
Molecular cloning workflow wherein this happens:
- T4 DNA Ligase
- PEG
- Vector to insert ratio
- Reaction time and temperature
- Purification/heat-inactivation (optional)
Ligation
Molecular cloning workflow wherein this happens:
- Transformation efficiencies
- Competent cell choices
- Chemical and electrocompetent cells
Transformation
Molecular cloning workflow wherein this happens:
- Blue/white colonies
- Positive selection
- Restriction digestion
- Colony PCR
- Sanger sequencing
Colony screening
Protein Analysis Workflow
- Lysate/Extracts ->
- Pour gel and assemble device ->
- Tank, Electrode Chamber, Gel cassette - Pipet samples and buffer ->
- Electrophoresis ->
- Separated protein bands
Another protein analysis workflow from new protein
- New protein ->
- Homology-Search ->
- Alignment ->
- Phylogenetic tree
Cellular Imaging/Visualization
- Fluorescence Microscopy
- Confocal Laser Scanning Microscopy
Prokaryotic Model Systems:
Bacteria (Escherichia coli)
Eukaryotic Model Systems: (and example study
- Yeast (Saccharomyces cerevisiae)
- Akt Protein Kinase Signaling - Worm (Caenorhabditis elegans)
- Evolution of Hox gene clusters - Zebrafish (Danio rerio)
- Cell Cycle Progression - Fruit Fly (Drosophila melanogaster)
- Neural Circuits for Innate Behavior - Mice (Mus musculus)
- Obesity, Cancer
- Alzheimer’s disease, anesthetics, AIDS & HIV - Plant (Arabidopsis thaliana)
- Casparian Strip (CS) gene expression
Some nobel prize using mice
Yellow fever vaccine
Discovery of penicillin
Structure of Nervous System
How many % is mice genes comparable to humans
98%
Attributes of A. thaliana as a model system
- Small size - requiring less growth space
- Shorter generation time - increasing the pace of research
- Large progeny for genetic analysis
- Small genome size (125Mb) - completely sequenced
- Small number of chromosomes (n=5)
- Amenable to transformation
- Spectrum of genetic and molecular resources
“Other” Routine Workflow in a Molecular Biology Lab
- Calculations
- Pipetting
- Weighing
- Culture Media Prep
- Chemicals/Solutions/Buffers Prep
- Measuring pH (pH meter not litmus paper!)
the number of moles of solute dissolved in one liter of solution
Molarity (mol/liter)
C1V1 = C2V2 ( or M1V1 = M2V2 )
C1: Initial concentration of the stock solution.
V1: Volume of the stock solution to be used.
C2: Final concentration of the diluted solution.
V2: Final volume of the diluted solution.
M1 and M2 are equivalent to C1 and C2: and represent molarity.
refers to the mass of solute (in grams) per 100 mL of solution
% w/v (weight/volume)
indicates the volume of solute (in mL) per 100 mL of solution.
% v/v (volume/volume)
Molecular Biology Lab Equipment
PCR Thermocycler
UV-Vis Spectrophotometer
Gel Documentation System
Electrophoresis Set-Ups
Lyophilizer
Refrigerated Centrifuge
equipment used for preservation by removing water from perishable materials through a process called freeze-drying
Lyophilizer
What happens to the DNA if there is repeated thawing and freezing of samples
reduced DNA size
how much does “Strictosidine-β-D-glucosidase” (SGD) cost?
Php 520.00/gram
For isolation of genomic, mitochondrial, bacterial, parasite or viral DNA
QIAamp DNA Mini Kit
are single-use tubes made from polypropylene for preparing, mixing, centrifuging, transporting and storing solid and liquid samples and reagents
Eppendorf tubes
Different freezers and fridges
-20 degrees
4 degrees
-80 degrees
a lab instrument that allows for quick and easy quantification of DNA, RNA, and protein samples using a small volume (1-2 µL) of sample, eliminating the need for cuvettes or capillaries.
NanoDrop spectrophotometer
a mass of unorganized, undifferentiated parenchyma cells that forms over a wound or when plant cells are cultured in vitro.
Callus
a powerful analytical technique used to separate, identify, and quantify volatile and semi-volatile compounds in complex mixtures, offering high sensitivity and specificity for various applications.
flavor and aroma
GC-MS (Gas Chromatography-Mass Spectrometry)
To accurately pipette relatively small volumes, typically 1-25 mL during experimentation, we use ________
Serological pipettes
Instrument used to measure and extract very small amounts of liquids from a solution
Often used to handle small amounts of solutions typical in molecular biology
Micropipettes
Disposable, autoclavable tips used to protect the micropipetter from contamination.
Used and disposed after EACH use in order to prevent contamination and reduce clean up and sterilization.
Plastic micropipette tips
Removes microorganisms (viruses, bacteria, fungi, etc.) and spores using high pressure and high temperature steam sterilization.
Autoclave
- Produce light of desired wavelength and it passes through your sample and reaches the photometer that measures its intensity.
- It reports the value as the amount of light absorbed (Absorbance (ABS).
Spectrometer/Spectrophotometer
Uses of spectrometer/spectrophotometer
- measure the growth of microorganisms like bacteria and yeast or other cells grown in suspension
- Find the concentration of proteins or nucleic acids
- Detect contaminants
Is an apparatus that rotates at high speed and separates substances of different densities.
Centrifuge
Uses of centrifuge:
- Remove cell elements from blood to provide cell-free serum or plasma.
- Separate subcellular components e.g. mitochondria, nuclei, etc.
- Remove insoluble matter from extracted proteins.
- Pellet DNA, RNA, protein, or lipids in isolation of macromolecules.
Tapered, specially-engineered test tubes for centrifuge work, designed to resist the high G-forces induced by centrifugal forces.
Microcentrifuge tubes
Stirs at controlled speeds to mix liquids
Vortex
Uses of vortex:
- Mix cells in suspensions.
- Mix reagents of an assay to create homogeneous solutions.
Container that uses water to heat or maintain a constant temperature in a highly controlled manner.
Water bath
Uses of water bath:
- To heat or thaw fragile biological substances like mammalian cells.
- To maintain more homogenous temperature control when working with nucleic acids and proteins.
Used to mix or agitate solutions, often prior to placement in the centrifuge and/or spectrophotometer
Shaker
Compound vs. Inverted Microscopes
Compound Light Microscope
- Uses a light source at the base in combination with various lenses to magnify images typically from 40x - 1000x
Inverted Microscope
- Uses a light source in the top along with various lenses in the base to magnify objects from 40x - 1000x
Sealed chamber with controls to manage temperature/sometimes humidity and CO2 levels.
Used to culture adherent cells under specific conditions mimicking normal or diseased in vivo environment.
Water Jacketed CO2 Incubator
Type of glass or plastic shallow round dish/flask or a coverslip usually coated with extracellular matrix (ECM)-like material
Petri Dishes/Flasks
Used to isolate DNA or protein fragments by size in a gel made of Agarose (DNA) or Polyacrylamide (Protein)
The electrophoresis chamber has an electrical charge running through it with the help of a power source to carry the DNA and protein through the gel.
Gel Electrophoresis Set-Up
DNA extraction by Phenol/Chloroform:
- Add **lysis buffer **into cell pellet and resuspend cells.
- Add RNase.
- Incubate at 37°C for 1 hour.
- Add proteinase K.
- Incubate at 50°C overnight.
- Add Phenol/Chloroform/Isoamylalcohol.
- Mix well.
- Centrifuge at 13 000 rpm for 10 minutes.
- Pipet aqueous phase into a new tube.
- Repeat from Step 6 to Step 9 for 2 times.
- Add 1/10th volume of 3M NaOAc.
- Add 2 volumes of 100% EtOH.
- The DNA start to precipitate immediately after shaking.
- Precipitate DNA up to 1 day at -70°C.
- Centrifuge at 13 000 rpm for 30 minutes at 4°C.
- Remove supernatant.
- Wash by 70% EtOH.
- Centrifuge at 13 000 rpm for 10 minutes.
- Remove supernatant.
- Remove remaining EtOH by pipeting.
- Air dry.
- Dissolve DNA pellet in appropriate volume of TE buffer.
What buffer is commonly used in molecular biology to store DNA and protect it from degradation by nucleases?
TE Buffer
TE buffer, or Tris-EDTA buffer, is a common solution in molecular biology used for nucleic acid storage, purification, and other procedures, containing Tris for pH buffering and EDTA for metal chelation, protecting DNA and RNA from degradation.
When plasma DNA is purified on a small scale, it is called a ____________:
Bacteria containing plasmid will be grown in liquid culture. The plasmids will then be extracted using alkaline lysis method. Finally, column chromatography will be performed to purify the plasmid DNA
Miniprep
Steps in performing alkaline lysis miniprep:
- Transfer a single colony of bacteria from a petri plate to a test tube with nutrient broth.
- Place the test tube in a shaking incubator and grow the culture overnight at 37°C (culture should appear cloudy after overnight).
- Transfer culture into 2 microcentrifuge tubes.
- Place the tubes in a microcentrifuge with the hinges facing outward. Centrifuge at top speed for 1 min.
- Remove the supernatant using a micropipette.
- Pipette resuspension solution into each tube.
- Resuspend the pellet by vortexing.
- Pipette lysis solution into each tube.
- Mix tubes by inverting (do not vortex).
- Pipette neutralization solution into each tube.
- Mix by inverting (a precipitate should form).
- Microcentrifuge for 5 mins at top speed.
- Place a mini column into each of the capless collection tubes.
- Transfer the supernatant into the mini column.
- **Microcentrifuge **the collection tubes with mini column for 1 min at top speed.
- Discard the liquid.
- Pipette wash solution into each column.
18.** Microcentrifuge** the columns for 1 min at top speed. - Discard liquid.
- Place the collection tubes in the microcentrifuge for 1 min to dry out.
- Discard the collection tubes and place the column into microcentrifuge tubes.
- Pipette elution solution onto the column bed of each column and leave for 1 min.
- Microcentrifuge for 1 min at top speed.
- Discard the columns.
- DNA is ready to use or can be stored at 4°C.
a solvent used to extract or remove a substance (like an antibody or DNA) that is adsorbed or bound to a solid material
Elution solution
The wells of the gel must be closest to which electrode
Negative (black) electrode = cathode
What is the electrical charge of DNA
Negative
In what direction will the DNA migrate in a gel electrophoresis
From cathode (black) to anode (red)
negative to positive
Where do we place the agarose gel?
Gel chamber
Steps in performing gel electrophoresis:
- Align the gel so the wells are closest to the negative electrode.
- Place the agarose gel into the gel chamber.
- Add electrophoresis running buffer to the reservoirs at each end of the gel chamber until the wells in the gel are covered by at least 2 mm of buffer.
- Place the samples to be loaded according to the lanes they are assigned to be running.
- Pipette the DNA sample (keep the pipette tip perpendicular to the row of wells to reduce the risk of accidentally puncturing the wells).
- Once all the samples have been loaded, avoid any bumping or movement in the gel chamber so the samples don’t spill to the adjacent wells.
- Place the lid on the gel chamber with the terminals correctly positioned to the matching electrodes on the gel chamber.
- Connect the electrodes to the power supply making sure that the electrodes match the terminals on the power supply.
- Switch the power supply on then set the correct voltage for running the samples.
- Set a clock for the proper time on your run.
- Press the start button to begin the flow of current that will separate the DNA fragments (at this point, bubbles are visible from the wires at each end of the gel box).
- In a few minutes, the samples will begin to migrate.
Which has more bubbles in the electrodes in a gel electrophoresis?
Negative (black) electrode
Materials needed for PCR
Sterile water
PCR buffer
dNTP mix
Forward primer
Reverse primer
DMSO (Option)
Taq DNA Polymerase
In PCR, ___________ enhances amplification, especially of GC-rich DNA, by inhibiting secondary structures in the template or primers, improving primer annealing and increasing the specificity and yield of the reaction.
dimethyl sulfoxide (DMSO)
Process of performing PCR:
- Mix PCR reagents on ice.
- Vortex gently.
- Aliquot mix into well plate.
- Apply DNA sample.
- Place plate in a PCR machine and run the program.
- Add DNA loading dye into each well
- Load PCR sample into gel well.
- Load DNA ladder.
- Electrophoresis.
- UV illumination.
Laboratory apparatuses in B3:
- Thermocycler (Thermal cycler) = Bio-rad
- Vortex mixer
- Centrifuge
- Water bath
- Gel doc = kinovett
- UV-Vis Spectrophotometer = thermoscientific genesys 150
- AGE = Mupid-exu
Agar = __________ + ___________
Agar + Agaropectin
Cell Lysis
- To break open the cell membrane and release
DNA. - Detergents (e.g., SDS, Triton X-100) dissolve lipid
membranes. - Physical disruption (e.g., grinding or sonication)
can also aid in breaking the cells open. - Nuclear membrane is also disrupted to release
nuclear DNA.
Removal of Proteins and Contaminants
- To eliminate proteins and other substances
that could contaminate the DNA. - Protease (e.g., proteinase K) breaks down
proteins, including histones. - Salts (e.g., NaCl) neutralize DNA’s negative charge
and help separate proteins. - Organic solvents may be used to remove lipids and
other contaminants.
DNA Precipitation
- To separate DNA from the solution.
- Adding alcohol (e.g., ethanol or isopropanol)
precipitates DNA by reducing its solubility. - DNA forms a solid pellet that can be collected via
centrifugation. - The alcohol causes DNA to aggregate into visible
strands.
