Gen Bio Lab Final Exam

what goes in the glass waste?

items that could cut or puncture the skin or trash bags.

• ex. pasteur pipettes, slides and slide covers, broken beakers

What goes in the sharps disposal?

items that present a significant threat to health and safety.

• ex. needles, scalpels, razor blades

what goes in the biohazard waste?

items that have contacted a possible biohazard like bacteria, recombinant nucleic acids like plasmids and vectors

• pipette tips, bacterial plates, microcentrifuge tubes

what goes in the general waste?

items that are neither sharp nor contaminated

metric system for volume?

liter

metric system unit for mass?

gram

metric system unit for temperature?

celcius

nanometer?

10^-9

how many pounds is 1000 g?

2.2

how many gallons is 1 L?

0.26

kilo?

10³

hecto?

10²

deca?

10¹

deci?

10^-1

centi?

10^-2

milli?

10^-3

micro?

10^-6

nano?

10^-9

pico?

10^-12

equation to make a solution?

how to calculate dilution?

C₁V₁=C₂V₂

equation to make solutions by mass? (also how to calculate mass from concentration)

C = m/V

• m= mass

• V= volume

• C = concentration

equation for preparing molar solutions?

n = C x V

• n= number of moles

serial dilution equation?

C₂ = C₁ x dilution factor

max and min of a p10 pipette?

0.5-10 μL

max and min of a p20 pipette?

2-20 μL

max and min of a p200?

20-200 μL

max and min of a p1000?

100-1000 μL

what does the eyepiece or ocular lense?

the lenses at the top that you look through. They are usually 10X or 15X power.

what is the arm?

Supports the tube and connects it to the base. It is used along with
the base to carry the microscope

what is the base?

The bottom of the microscope, used for support.

what is the illuminator?

Light source found inside the illuminator housing, a steady
light source used in place of a mirror in modern microscopes.

what is the stage?

The flat platform where you place your slides. Stage clips hold the
slides in place.

what is the stage adjustment knob?

Moves the stage in the x and y directions. This allows you to smoothly and slowly move the slide as you’re looking through the ocular lenses

what is the objective lenses?

Usually, you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) ocular lens, we get total magnifications of 40X (4X times 10X), 100X, 400X and 1000X. The shortest lens is the lowest power, the longest one is the lens with the greatest power. The high-power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide

what is the condenser?

The condenser gathers light from the microscope light source and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire view field.

what is the iris diaphgram?

The iris diaphragm on our microscopes is part of the condenser. You’ll see a tab that you can move back, this controls the iris diaphragm. The iris diaphragm has different-sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. In our model placement for using the different objective lenses and phase contrast (PH) is indicated on the condenser. However, there is no set rule regarding which setting to use for a particular objective lens. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire, and the particular objective lens in use. Most of the time the setting indicated on the condenser should work well, but sometimes you might move the iris diaphragm off that mark and get a better image. However, when using phase contrast, you should always have the iris diaphragm set to PH.

what is the objective nosepiece?

Also called the rotating nosepiece, this is the part that holds two or more objective lenses and can be rotated to easily change magnifying power.

what is the coarse focusing knob?

This is used to focus the microscope. It is always whused first, and it is used only with the low power objective, in our case 4x

what is the fine focusing knob?

This is used to focus the microscope. It is used with the high power objectives to bring the specimen into better focus.

what is chlamydomonas reinhardtii?

single-celled, biflagellate, green alga, approximately 10μ long. The genus Chlamydomonas contains several species that have become popular as research tools, but by far the most frequently used and the species we will be discussing is Chlamydomonas reinhardtii

why are chlamydomonas useful?

simple and cheap to grow, can be handled by standard microbial techniques, is motile, and shows easily measured behavioral trait

what is the independent variable?

the factor being manipulated

what is the dependent variable?

the factor thought to be impacted by the manipulation

what is a positive control?

a control group that is treated with a known experimental factor to produce a known effect.

what is the negative control?

a control group that is not exposed to the experimental treatment and should produce no effect

what is the role of statistics?

all data exhibit variability, and it is the role of statistics to quantify this variability and allow scientists to make more accurate statements about their data

what is descriptive statistics?

sum up major attributes of a dataset using measures such as the mean, median, and standard deviation

what is inferential statistics?

look at patterns in data make judgments about data, identify relationships between variables in datasets, and make inferences about larger populations based on smaller samples of data

what does standard deviation tell us?

a measure of how dispersed the data is in relation to the mean. Low standard deviation means data are clustered around the mean, and high standard deviation indicates data are more spread out.

why use a standard curve?

a type of graph used as a quantitative research technique. Multiple samples with known properties are measured, which then allows the same properties to be determined for unknown samples using a graph. The samples with known properties are the standards, as the graph is the standard curve.

what is the initial rate of reaction?

the region where the amount of product is formed increases in a linear fashion

• equal to the slope of the line and is the m in y=mx + b

what biotechniques were used for the DNA cloning project in order?

DNA extraction, Gel electrophoresis, restriction digest, PCR purification, ligation, transformation, DNA sequencing

• also DNA quantifies after some steps

what is a vector and what was our vector?

circular pieces of DNA that are outside of the bacterial chromosome. Ours is a bacterial plasmid pET-41a

what is step 1 of a PCR and what is done?

denaturation: the reaction is heated to 95 degrees celsius and double stranded DNA is separated into single strands as hydrogen bonds break

what is step 2 of PCR?

hybridization/annealing: reaction temperature reduced to between 55 and 65 degrees to allow primer annealing

what is step 3 of PCR?

extension: temperature is raised to 72 degrees, and Taq synthesizes DNA

what is an insert? what was our insert?

a segment of DNA that is inserted into a vector. Usually contains the gene of interest. Ours is eGFP the jellyfish gene for enhanced fluorescent proteins.

what is gel electrophoresis?

a widely used technique for the analysis of nucleic acids and proteins. Usually uses agarose gel. Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. we used it to determine the presence and size of PCR product and vector.

what is commonly used to visualize DNA?

ethidium bromide

what is the function of loading dye?

helps the sample sink into the well and allows us to estimate the progress of the DNA across the gel.

what are the 2 restriction enzymes we used?

NotI and Ncol

how do restriction enzymes work?

each restriction enzyme will only cut DNA at a specific DNA sequence.

why did we digest both the vector and the insert with the same restriction enzymes?

we need the vector and insert to have complementary sticky ends

why do we need to clean up the DNA after PCR or restriction digest?

we only want DNA

what does DNA ligase do?

create phosphodiester bonds in a 2 step process

why did we put IPTG in our plates?

when IPTG is present, it allows RNA polymerase to move through the lac operator and transcribe the GFP gene we placed in the MCS

why did we need the 45 minute recovery incubation?

It takes time for the protein to be expressed (i.e., the DNA transcribed into mRNA and translated into protein) and if the bacteria doesn’t have the aminoglycoside phosphotransferase protein it will die when it comes into contract with Kanamycin.

how many bp is the insert?

734

how many bp is the vector?

5933

what do ddNTP’s do?

randomly terminate PCR reactions

what are some uses for DNA cloning?

producing recombinant proteins, gene therapy, cloning for functional studies, creating genetically modified organisms (GMO’s), and vaccine development

what is a spectrophotometer?

a device used to measure the amount of light that a sample absorbs at different wavelengths. It works by passing light through a sample and measuring how much light is absorbed or transmitted by the sample.

how to correctly load and read a cuvette in a spectrophotometer?

1. before use make sure the cuvette is clean, clean with kim wipe

2. fill cuvette with sample

3. blank the spectrophotometer

4. align the sample cuvette correctly, the flat clear sides should be orientated to face the light beam

5. measure the absorbance

what does ABS repersent?

measure of the amount of light absrobed by a sample at a specfifc wavelenght.

why is wavelength important?

it determines which part of the electromagnetic spectrum the light is coming from, and different substances absorb light at different wave lengths.

how does DNA extraction work?

the cells are broken open using a detergent or other chemicals to dissolve the membrane, releasing the DNA into a solution. Next, proteins and other cellular debris are removed by adding enzymes or salts that help separate them from the DNA. The DNA is then precipitated out of the solution by adding alcohol, such as ethanol or isopropanol, which makes the DNA clump together. Finally, the DNA is washed and dissolved in a buffer.

what does the silica membrane do in DNA extraction?

helps only DNA bind to the membrane. Once DNA is bound to the silica membrane, other contaminants can be washed away. The DNA can be eluted from the membrane after the other components have been washed away.

Using a nano spectrophotometer to quantify DNA, what is the 230 nm tell us?

typically used to detect contaminants, especially organic compounds like phenol, guanidine, or other chemicals used in the extraction process. These substances absorb light around this wavelength.

Using a nano spectrophotometer to quantify DNA, what is the 260 nm tell us?

primary wavelength used to measure the concentration of nucleic acids (both DNA and RNA) because DNA absorbs strongly at this wavelength.

Using a nano spectrophotometer to quantify DNA, what is the 280 nm tell us?

primarily used to detect protein contamination. Proteins absorb light at this wavelength, particularly due to the aromatic amino acids like tryptophan and tyrosine.

Using a nano spectrophotometer to quantify DNA, what is the 260/280 nm tell us?

indication of protein contamination

Using a nano spectrophotometer to quantify DNA, what is the 260/230 nm tell us?

ratio helps to detect contamination by organic solvents or other impurities from the DNA extraction process

how does gel electrophoresis work?

1. gel preparation

2. sample loading

3. electrophoresis process

4. separation by size

5. staining

what does gel electrophoresis accomplish?

separation of DNA fragments by size available for analysis. Also purity and integrity check

how to read DNA gels?

compare band pattern to a ladder

how does nucleic acid clean up work?

1. DNA is mixed with a buffer that helps nucleic acids bind to the silica membrane

2. the column is then washed with one or more buffers to remove contaminants

3. elution: the nucleic acids are eluted by adding an elution buffer, this releases the purified DNA into solution ready for further analysis

what does nucleic acid clean up accomplish?

removal of contaminants, purification, and makes DNA concentrated

how do restriction enzymes work?

proteins that recognize and cut DNA at specific sequences called recognition sites. Recognition sites are short palindromic sequences. Can result in blunt or sticky ends.

what are common restriction enzymes?

EcoRI, HindIII, BamHI, and SmaI

where do restriction enzymes come from?

they are apart of a bacteria’s immune system, protecting against invading viral DNA (bacteriophages). Bacteria use them to cut and inactivate foreign DNA

what do restriction enzymes accomplish?

critical for DNA cloning, gene editing, analysis, and genotyping

How does PCR work?

a technique used to amplify a specific DNA sequence. Works by cycling through a series of temperature changes that enable the DNA to be replicated

what enzyme is involved in PCR and where did it come from?

Taq polymerase it is isolated from the bacteria Thermus aquaticus and it can withstand high temperatures.

what does PCR accomplish?

DNA amplification

How do ligation reactions work?

used to join DNA fragments by forming covalent bonds between the phosphate group of 1 DNA strand and the hydroxyl group of another. DNA fragments with compatible ends (sticky) are mixed with ligation buffer and ligase.

What enzyme is involved in ligase reaction and where did it come from?

T4 DNA ligase, derived from T4 bacteriophage a virus that infects E.coli

What does Ligation reactions accomplish?

ligating 2 complementary DNA together.

How does transformation work?

involves introducing foreign DNA into a host cell, such as bacteria to modify its genetic material. Done by mixing plasmid DNA in competent cells, heat shocking to open the membrane and allowing DNA to enter, cells are then incubated to recover, and then put on agar plates containing an antibiotic.

What does transformation accomplish?

allows you to genetically modify cells