Introduction to Cell and Molecular Biology Laboratory

Question 1

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)

Answer 1

1273 aa

Question 2

_________ visually form a characteristic bulbous, crown-like halo surrounding the viral particle

Answer 2

S protein trimers

Question 3

are an important class of secondary metabolites known for their protection agaisnt UV radiation and other stress factors

Answer 3

Mycrosporine-like amino acids (MAAs)

Question 4

Cyanobacteria produce a variety of MAAs, including ________, the active ingredient in many sunscreen creams

Answer 4

Shinorine

Question 5

Topics under cell and molecular biology

Answer 5

1. Molecular mechanisms inside the cell.
2. Flow of information within and between cells.
3. Intracellular and transmembrane transport.
4. Control of gene expressions.
5. Survival and death of cells.
6. Formation of cancerous cells.

Question 6

Course Learning Objectives

After finishing this course, you should be able to have:

Answer 6

• 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.

Question 7

Routine Workflow in a Molecular Biology Lab

Answer 7

1. Collecting samples, preparing samples
2. Extracting DNA/RNA/proteins
3. Determining DNA/RNA/protein concentration
4. Amplifying gene of interest (GOI) by Polymerase Chain Reaction (PCR)
5. Detecting DNA/RNA/protein by gel electrophoresis
6. Documenting gel images (“Gel Doc”)
7. Downstream analysis: DNA sequencing or cloning, etc.

Question 8

DNA Workflow (arrows)

Answer 8

1. DNA Extraction
2. Pure DNA
3. (AGE/Spectrophotometry = DNA Check: Purity, Quality, Quantity)/PCR
4. From PCR -> AGE/Spectrophotometry
5. From AGE -> PCR product purification
6. Sequencing, cloning, other applications

Question 9

DNA Analysis Workflow

Answer 9

DNA Fingerprinting/DNA Barcoding
- Multiple Sequence Alignment ->
- Phylogenetic Tree

Question 10

RNA Analysis Workflow

Answer 10

Gene Expression Profiling

1. Cell culture
2. Cell lysis/RNA Isolation
3. Reverse transcription
4. Quantitative PCR (qPCR)

Question 11

a technique that amplifies and simultaneously detects the amplification product of nucleic acids, allowing for the quantification of DNA or RNA

Answer 11

Quantitative PCR (qPCR)

Question 12

The Molecular Cloning Workflow

Answer 12

Vector Preparation -> Insert Preparation -> Ligation -> Transformation -> Colony Screening

Gene from a plasmid/DNA fragment is ligated into a vector (another plasmid) = Recombinant DNA technology

Question 13

bacteria carrying both the WT gene and the integrated plasmid

Answer 13

merodiploid state

Question 14

Molecular cloning workflow wherein this happens:

• Restriction digestion
• Dephosphorylation
• Blunt-end creation (optional)
• Purification

Answer 14

Vector Preparation

Question 15

Molecular cloning workflow wherein this happens:

• Restriction Digestion
• Blunt-end creation (optional)
• Purification

Answer 15

Insert Preparation

Question 16

Molecular cloning workflow wherein this happens:

• T4 DNA Ligase
• PEG
• Vector to insert ratio
• Reaction time and temperature
• Purification/heat-inactivation (optional)

Answer 16

Ligation

Question 17

Molecular cloning workflow wherein this happens:

• Transformation efficiencies
• Competent cell choices
• Chemical and electrocompetent cells

Answer 17

Transformation

Question 18

Molecular cloning workflow wherein this happens:

• Blue/white colonies
• Positive selection
• Restriction digestion
• Colony PCR
• Sanger sequencing

Answer 18

Colony screening

Question 19

Protein Analysis Workflow

Answer 19

1. Lysate/Extracts ->
2. Pour gel and assemble device ->
   - Tank, Electrode Chamber, Gel cassette
3. Pipet samples and buffer ->
4. Electrophoresis ->
5. Separated protein bands

Question 20

Another protein analysis workflow from new protein

Answer 20

1. New protein ->
2. Homology-Search ->
3. Alignment ->
4. Phylogenetic tree

Question 21

Cellular Imaging/Visualization

Answer 21

1. Fluorescence Microscopy
2. Confocal Laser Scanning Microscopy

Question 22

Prokaryotic Model Systems:

Answer 22

Bacteria (Escherichia coli)

Question 23

Eukaryotic Model Systems: (and example study

Answer 23

1. Yeast (Saccharomyces cerevisiae)
   - Akt Protein Kinase Signaling
2. Worm (Caenorhabditis elegans)
   - Evolution of Hox gene clusters
3. Zebrafish (Danio rerio)
   - Cell Cycle Progression
4. Fruit Fly (Drosophila melanogaster)
   - Neural Circuits for Innate Behavior
5. Mice (Mus musculus)
   - Obesity, Cancer
   - Alzheimer’s disease, anesthetics, AIDS & HIV
6. Plant (Arabidopsis thaliana)
   - Casparian Strip (CS) gene expression

Question 24

Some nobel prize using mice

Answer 24

Yellow fever vaccine
Discovery of penicillin
Structure of Nervous System

Question 25

How many % is mice genes comparable to humans

Answer 25

98%

Question 26

Attributes of A. thaliana as a model system

Answer 26

- 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

Question 27

“Other” Routine Workflow in a Molecular Biology Lab

Answer 27

* Calculations * Pipetting * Weighing * Culture Media Prep * Chemicals/Solutions/Buffers Prep * Measuring pH (pH meter not litmus paper!)

Question 28

the number of moles of solute dissolved in one liter of solution

Answer 28

Molarity (mol/liter)

Question 29

C1V1 = C2V2 ( or M1V1 = M2V2 )

Answer 29

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.

Question 30

refers to the mass of solute (in grams) per 100 mL of solution

Answer 30

% w/v (weight/volume)

Question 31

indicates the volume of solute (in mL) per 100 mL of solution.

Answer 31

% v/v (volume/volume)

Question 32

Molecular Biology Lab Equipment

Answer 32

PCR Thermocycler UV-Vis Spectrophotometer Gel Documentation System Electrophoresis Set-Ups Lyophilizer Refrigerated Centrifuge

Question 33

equipment used for preservation by removing water from perishable materials through a process called freeze-drying

Answer 33

Lyophilizer

Question 34

What happens to the DNA if there is repeated thawing and freezing of samples

Answer 34

reduced DNA size

Question 35

how much does "Strictosidine-β-D-glucosidase" (SGD) cost?

Answer 35

Php 520.00/gram

Question 36

For isolation of genomic, mitochondrial, bacterial, parasite or viral DNA

Answer 36

QIAamp DNA Mini Kit

Question 37

are single-use tubes made from polypropylene for preparing, mixing, centrifuging, transporting and storing solid and liquid samples and reagents

Answer 37

Eppendorf tubes

Question 38

Different freezers and fridges

Answer 38

-20 degrees 4 degrees -80 degrees

Question 39

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.

Answer 39

NanoDrop spectrophotometer

Question 40

a mass of unorganized, undifferentiated parenchyma cells that forms over a wound or when plant cells are cultured in vitro.

Answer 40

Callus

Question 41

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

Answer 41

GC-MS (Gas Chromatography-Mass Spectrometry)

Question 42

To accurately pipette relatively small volumes, typically 1-25 mL during experimentation, we use ________

Answer 42

Serological pipettes

Question 43

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

Answer 43

Micropipettes

Question 44

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.

Answer 44

Plastic micropipette tips

Question 45

Removes microorganisms (viruses, bacteria, fungi, etc.) and spores using high pressure and high temperature steam sterilization.

Answer 45

Autoclave

Question 46

- 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).

Answer 46

Spectrometer/Spectrophotometer

Question 47

Uses of spectrometer/spectrophotometer

Answer 47

- 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

Question 48

Is an apparatus that rotates at high speed and separates substances of different densities.

Answer 48

Centrifuge

Question 49

Uses of centrifuge:

Answer 49

- 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.

Question 50

Tapered, specially-engineered test tubes for centrifuge work, designed to resist the high G-forces induced by centrifugal forces.

Answer 50

Microcentrifuge tubes

Question 51

Stirs at controlled speeds to mix liquids

Answer 51

Vortex

Question 52

Uses of vortex:

Answer 52

- Mix cells in suspensions. - Mix reagents of an assay to create homogeneous solutions.

Question 53

Container that uses water to heat or maintain a constant temperature in a highly controlled manner.

Answer 53

Water bath

Question 54

Uses of water bath:

Answer 54

- To heat or thaw fragile biological substances like mammalian cells. - To maintain more homogenous temperature control when working with nucleic acids and proteins.

Question 55

Used to mix or agitate solutions, often prior to placement in the centrifuge and/or spectrophotometer

Answer 55

Shaker

Question 56

Compound vs. Inverted Microscopes

Answer 56

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

Question 57

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.

Answer 57

Water Jacketed CO2 Incubator

Question 58

Type of glass or plastic shallow round dish/flask or a coverslip usually coated with extracellular matrix (ECM)-like material

Answer 58

Petri Dishes/Flasks

Question 59

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.

Answer 59

Gel Electrophoresis Set-Up

Question 60

DNA extraction by Phenol/Chloroform: 1. Add **lysis buffer **into cell pellet and resuspend cells. 2. Add **RNase**. 3. Incubate at 37°C for 1 hour. 4. Add **proteinase K**. 5. Incubate at 50°C overnight. 6. Add **Phenol**/Chloroform/Isoamylalcohol. 7. Mix well. 8. **Centrifuge** at 13 000 rpm for 10 minutes. 9. Pipet aqueous phase into a new tube. 10. **Repeat** from Step 6 to Step 9 for 2 times. 11. Add 1/10th volume of 3M NaOAc. 12. Add 2 volumes of **100% EtOH.** 13. The DNA start to precipitate immediately after shaking. 14. Precipitate DNA up to 1 day at -70°C. 15. **Centrifuge** at 13 000 rpm for 30 minutes at 4°C. 16. **Remove supernatant**. 17. **Wash by 70% EtOH**. 18. **Centrifuge** at 13 000 rpm for 10 minutes. 19. **Remove supernatant.** 20. Remove remaining EtOH by pipeting. 21. Air dry. 22. Dissolve DNA pellet in appropriate volume of **TE buffer**.

Question 61

What buffer is commonly used in molecular biology to store DNA and protect it from degradation by nucleases?

Answer 61

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.

Question 62

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

Answer 62

Miniprep

Question 63

Steps in performing alkaline lysis miniprep: 1. Transfer a single colony of bacteria from a petri plate to a **test tube with nutrient broth**. 2. Place the test tube in a **shaking incubator** and grow the culture overnight at 37°C (culture should appear cloudy after overnight). 3. **Transfer culture into 2 microcentrifuge tubes**. 4. Place the tubes in a **microcentrifuge** with the hinges facing outward. Centrifuge at top speed for 1 min. 5. **Remove the supernatant** using a micropipette. 6. Pipette **resuspension solution** into each tube. 7. Resuspend the pellet by **vortex**ing. 8. Pipette **lysis solution** into each tube. 9. **Mix tubes by inverting** (do not vortex). 10. **Pipette neutralization solution** into each tube. 11. **Mix by inverting** (a precipitate should form). 12. **Microcentrifuge** for 5 mins at top speed. 13. Place a mini column into each of the capless collection tubes. 14. **Transfer the supernatant into the mini column**. 15. **Microcentrifuge **the collection tubes with mini column for 1 min at top speed. 16. **Discard the liquid**. 17. Pipette **wash solution** into each column. 18.** Microcentrifuge** the columns for 1 min at top speed. 19. **Discard liquid**. 20. Place the collection tubes in the **microcentrifuge** for 1 min to dry out. 21. Discard the collection tubes and **place the column into microcentrifuge tubes**. 22. Pipette **elution solution** onto the column bed of each column and leave for 1 min. 23. **Microcentrifuge** for 1 min at top speed. 24. **Discard the columns**. 25. DNA is ready to use or can be stored at 4°C.

Question 64

a solvent used to extract or remove a substance (like an antibody or DNA) that is adsorbed or bound to a solid material

Answer 64

Elution solution

Question 65

The wells of the gel must be closest to which electrode

Answer 65

Negative (black) electrode = cathode

Question 66

What is the electrical charge of DNA

Answer 66

Negative

Question 67

In what direction will the DNA migrate in a gel electrophoresis

Answer 67

From cathode (black) to anode (red) negative to positive

Question 68

Where do we place the agarose gel?

Answer 68

Gel chamber

Question 69

Steps in performing gel electrophoresis: 1. Align the gel so the wells are closest to the negative electrode. 2. Place the agarose gel into the gel chamber. 3. 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. 4. Place the samples to be loaded according to the lanes they are assigned to be running. 5. Pipette the DNA sample (keep the pipette tip perpendicular to the row of wells to reduce the risk of accidentally puncturing the wells). 6. 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. 7. Place the lid on the gel chamber with the terminals correctly positioned to the matching electrodes on the gel chamber. 8. Connect the electrodes to the power supply making sure that the electrodes match the terminals on the power supply. 9. Switch the power supply on then set the correct voltage for running the samples. 10. Set a clock for the proper time on your run. 11. 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). 12. In a few minutes, the samples will begin to migrate.

Question 70

Which has more bubbles in the electrodes in a gel electrophoresis?

Answer 70

Negative (black) electrode

Question 71

Materials needed for PCR

Answer 71

Sterile water PCR buffer dNTP mix Forward primer Reverse primer DMSO (Option) Taq DNA Polymerase

Question 72

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.

Answer 72

dimethyl sulfoxide (DMSO)

Question 73

Process of performing PCR:

Answer 73

1. Mix PCR reagents on ice. 2. Vortex gently. 3. Aliquot mix into well plate. 4. Apply DNA sample. 5. Place plate in a PCR machine and run the program. 6. Add DNA loading dye into each well 7. Load PCR sample into gel well. 8. Load DNA ladder. 9. Electrophoresis. 10. UV illumination.

Question 74

Laboratory apparatuses in B3:

Answer 74

- Thermocycler (Thermal cycler) = Bio-rad - Vortex mixer - Centrifuge - Water bath - Gel doc = kinovett - UV-Vis Spectrophotometer = thermoscientific genesys 150 - AGE = Mupid-exu

Question 75

Agar = __________ + ___________

Answer 75

Agar + Agaropectin

Question 76

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.

Question 77

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.

Question 78

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.