Lab Exam Notes
Micropipettors
The proper procedure to use a micropipettor
Unlock it
How to set the volume and how to read the numbers in the volume window
Which micropipettor is appropriate for taking a given volume of liquid (maximum volume it can take is the name of the pipette) 1000 microliters is 1 liter
P10: For volumes between 0.5-10 uL
4 digit window; bottom two are decimal places
P20: For volumes between 2-20 µL.
Three digits window; bottom digit is a decimal place; 60 uL expressed as 06
P200: For volumes between 20-200 µL.
Three digits window; 060 corresponds to 60 uL (the tick marks are decimals)
P1000: For volumes between 100-1000 µL (1 L)
4 digit window; first digit should be 0 for all measurements except 1000
Why disposable tips must be used
Prevents contamination between samples
The difference between the first and the second stops on a micropipettor
First Stop: allows you to draw the correct volume of liquid
Second Stop: allows you to dispense the entire volume
Microscopy Lab
The difference between compound and dissecting microscopes
Compound Microscope- Used for viewing very small specimens can view things at higher magnification
Magnification: Provides higher magnification (typically 40x to 1000x or more).
Illumination: Light shines through the specimen (transmitted light).
Dissecting Microscope- Used for viewing larger specimens in 3D
Magnification: Provides lower magnification (typically 10x to 40x).
Illumination: Light is directed onto the specimen's surface (reflected light).
Recognize the major parts of the microscopes and their functions
Eyepiece (Ocular Lens): The lens you look through; typically provides 10x magnification.
Objective Lenses: Multiple lenses with varying magnifications (usually 4x, 10x, 40x, and 100x). They focus light on the specimen.
Stage: The platform where you place the specimen slide.
Stage Clips: Hold the slide in place on the stage.
Coarse Focus: Adjusts the focus for low-power objectives.
Fine Focus: Fine-tunes the focus for high-power objectives.
Condenser: Focuses light onto the specimen (found mostly on compound microscopes).
Diaphragm: Controls the amount of light reaching the specimen.
Illuminator: Provides the light source for the microscope.
Arm: Supports the body tube and connects it to the base.
Base: The bottom part of the microscope that supports the entire structure.
Lamp
Be able to describe how wet mount slides are prepared
Step 1: Place the Specimen: Place a small drop of liquid (e.g., water or staining solution) on a clean microscope slide.
Step 2: Add the Sample: Place the specimen (e.g., a leaf, small organism, or a sample from a pond water) into the drop of liquid.
Step 3: Apply a Coverslip: Carefully place a cover slip at an angle over the drop, avoiding air bubbles. Lower it gently to avoid trapping air bubbles.
Step 4: Examine: Place the slide on the microscope stage, adjust the light and focus, and examine the sample.
Know how magnification is calculated
Multiply by 10
The cell
Know the reason behind using stains such as Janus Green B, etc.
Purpose of Stains: Staining is used to enhance the contrast of transparent specimens and highlight specific cell structures or components (like the nucleus or mitochondria).
Janus Green B: A stain used to visualize mitochondria in cells, as it binds to mitochondria and highlights them by turning them a distinctive color (usually green or blue-green).
Other Common Stains:
Iodine: Stains starch in plant cells, making it useful for identifying starch storage in cells (e.g., in amyloplasts).
Methylene Blue: Stains the nucleus and cell structures.
Crystal Violet: Used for staining bacterial cells in gram-staining procedures.
Know the difference between Eukaryotic and Prokaryotic cells you observed under the microscope
Recognize chloroplasts, amyloplasts, the nucleus, and plant versus animal cells
Probably won't test this
Recognize the cells from the different species observed under the microscope
Probably wont test this
Fermentation Lab
The basic process
Why the yeast in your tubes resort to fermentation instead of aerobic respiration
Lack of oxygen
Yeast resorts to fermentation when oxygen is unavailable, as it cannot perform aerobic respiration. Fermentation allows glycolysis to continue by regenerating NAD+
Why alcohol fermentation is needed in yeast to carry out glycolysis
During glycolysis, glucose is converted into pyruvate, and NAD+ is reduced to NADH. For glycolysis to continue, NAD+ needs to be regenerated. In the absence of oxygen, alcohol fermentation allows the regeneration of NAD+ by converting pyruvate into ethanol and CO₂, thus allowing glycolysis to continue.
How efficient fermentation is
It is much less efficient than aerobic respiration and has a low energy yield
What amylase does to starch
It is an enzyme that breaks down starch, helps speed up the fermentation process
What gas is produced
Carbon Dioxide (CO2)
The experiment
How we take advantage of the gas produced to measure fermentation rates
The rate of reaction for fermentation can be determined by measuring CO2 concentrations
The effect temperature is expected to have on this system
At high temperature, enzymes responsible for this system can denature
At low temperatures, enzymes responsible will slow down, decreasing rate of reaction
Optimal temp is 37 degrees Celsius
The point of performing treatment with starch versus treatment with starch + amylase
Starch Only: If yeast is given starch but no amylase, it cannot break down the starch into smaller sugars. Yeast can only ferment sugars like glucose or maltose, so no or very little fermentation will occur with starch alone.
Starch + Amylase: When amylase is added to the starch, the enzyme will break the starch down into smaller sugars (like maltose or glucose), which yeast can then ferment.
Goal: This treatment ensures that starch is converted into fermentable sugars, allowing you to test how efficiently yeast can ferment the starch after it is broken down by amylase.
The hypotheses for the 3 treatments and the logic behind them
5 % Glucose and yeast will have the second highest rate of fermentation
Logic: glucose is already a simple sugar which can be fermented.
1 percent Starch and yeast , in the absence of amylase, will have the lowest rate of reaction
Starch is a polysaccharide meaning it cannot be fermented unless it is broken down into simple sugars. Yeast can only ferment simple sugars.
1 percent Starch, yeast and amylase, will have the highest rate of reaction
Amylase is an enzyme so it will speed up reaction rates.
Given the data, be able to graph the data and discuss whether the data match the experimental predictions
Not doing this
Starch prints
The logic behind the process
Based on the fact that after photosynthesis is conducted, excess glucose is deposited into the plant cells as starch. By using a chemical (iodine) that reacts with starch, an image can be created on a leaf providing an outline of where photosynthesis and starch production had occurred.
If plant in dark not going to do photosynthesis, when you remove the plant to the light areas of leaf that is exposed to the light will undergo photosynthesis. Those areas will eventually produce starch. Iodine can be used to stain starch. That iodine stain will stain in the pattern of a picture (only areas that are stained are the ones which starch that has undergone photosynthesis). First boiled it in water then boiled it in ethanol that stripped all of the chlorophyll green color.
How a negative image is turned into a positive image inside the leaf tissue 2
(positive image) Light will pass through this area in the beginning. It is the area that is exposed to light and will show a blue-black color, essentially printing darker after iodine staining
While areas kept in the dark will not pass light and will not be stained since there is no starch production there (negative image) this will lead to a lighter expression.
The leaf area exposed to light will conduct photosynthesis and under bright light intensity, starch will not be transported out of the leaf but will remain localized. Therefore, areas exposed to light will have starch stored there and when we stain the leaf with iodine, the area exposed to light will be stained a dark blue-black color, which inhibits photosynthesis, since dark color blocks out light. While the area not exposed to light (the negative image) will remain light in color. This means only the positive image will have dark areas (where starch is stored) and the negative image will have light areas reflective of the image. In terms of photosynthesis, the area that is dark will not pass light through, while the area that is light will pass through, ultimately they switch responsibilities.
Relationship between light levels and rate of photosynthesis / starch production
The rate of photosynthesis (and therefore starch production) is directly linked to light levels—higher light leads to more glucose production and more starch.
What the experiment tells us about transport of starch
Starch is produced during photosynthesis and can be transported out of the leaf but is generally localized; it stays where it is produced in the leaf during photosynthesis.
Probing Probiotics
What probiotics are
Probiotics are live microorganisms, often bacteria or yeast, that provide health benefits when consumed in adequate amounts.
Aid in healthy gut bacteria growth.
Selective plating: uses and limitations, how to interpret the growth on the plates
Selective plating- uses specific nutrients or chemicals to encourage the growth of certain microorganisms while inhibiting others.
The plates we used were MRS plates kinda orange. It selectively grew only lactobacilli bacteria and not ecoli bacteria. Ecoli can not grow on that plate that is called selective planting. If we had a normal plate like agar ecoli would have been able to grow.
What a bp (base pair) is
1 base pair is 1 nucleotide connected through hydrogen bonds
Also used as a measurement for the length of a DNA fragmen
How PCR works
amplifies specific DNA sequences
First we have to extract our dna then can use for pcr
Steps:
Denaturation: heat up samples so that the dna strands separate
Annealing: (the attaching step) The reaction is cooled, allowing primers (short DNA sequences) to bind to the complementary regions of the target DNA.
Replication: The DNA polymerase replicates DNA.
*ran cycle 40 times so we produced a lot of dna product
Significance of primers in relation to the target sequence
Ensure that the PCR reaction amplifies the target sequence and not random DNA sequences
Steps in the cycle
Denaturation: DNA is heated to around 94-98°C to break the hydrogen bonds between the two strands of the DNA template, creating single-stranded DNA.
Annealing: The temperature is lowered to about 50-65°C, allowing primers to bind (anneal) to the complementary sequences on the single-stranded DNA.
Extension: The temperature is raised to around 75-80°C for DNA polymerase to extend the primers and synthesize the new DNA strand.
What you must know before you can set up the PCR reaction
You need to decide the Locus that you want to amplify
Size of the segment you intend to amplify
Your positive and negative controls
Why a reference gene such as FtsZ would be included in your PCR reactions
Can help you figure out what is in your sample, didn't do this though because primers of dna did not work
Why water is used in one of the PCR reactions
Water is the negative control because there is no dna in water,
so no pcr content, so there should be no band in the water sample
What we need to do (in general terms) to extract DNA
Break apart the cell
Spin the samples
Because we don't want any of the organelles
What gel electrophoresis is and how it works
Gel Electrophoresis is a method used to separate DNA fragments based on their size.
Gel Preparation: A gel matrix (usually agarose) is prepared and loaded into an electrophoresis chamber.
DNA Loading: DNA samples (typically mixed with a dye) are loaded into wells at one end of the gel.
Electrical Current: An electrical current is applied across the gel. DNA molecules, which are negatively charged due to their phosphate backbone, will move towards the positive electrode.
Separation: Smaller DNA fragments move faster through the gel, while larger fragments move more slowly, resulting in separation by size.
The role of the electrical current
Positive electrodes that attracts dna and helps it separate out by size
If we didn't have electrocurrent all pieces of dna would be together won't be separated out so we won't see what's in it
The role of the gel material
Important because it is like a matrix
So smaller dna fragments will flow quicker, larger dna fragments will go slower
The role of the dye
Visual cue so you can see the sample on the gel
The role of the stain
A stain (such as ethidium bromide or SYBR Green) binds to the DNA and fluoresces under UV light, allowing the DNA fragments to be seen as bands in the gel.
How to find the size of the DNA fragments showing up on a gel
To determine the size of DNA fragments, a DNA ladder or marker (which contains DNA fragments of known sizes) is run alongside the samples. By comparing the migration distance of the unknown DNA fragments to the known fragments in the ladder, you can estimate the size of the DNA fragments in base pairs.
*The size that we were looking 129 base pairs
Why we sometimes see additional bands on the gel
Amplify more than one part of the dna
Primers are not very specific so it binds to multiple parts of dna and produces multiple products (dont want the additional banding)
Whether there were differences between the samples studied, based on your data: be able to answer this looking at a picture of a gel
How PCR and gel electrophoresis are used in forensic identification of genes or species
Can amplify human vs animal vs plant dna
Data Analysis
For a given scenario and data set be able to
Calculate means and standard deviations, given the formulas and simple calculators
Calculate t-values, given the formulas and simple calculators
Interpret t-tests and the confidence levels given the t table
Figure out whether you should accept or reject an alternative hypothesis based on the results of the test used
Paired t test- compares the means of the same group under two different conditions ( example pH)
Unpaired t test- compares the means of the different group (example phenylamines)
X= mean
n= sample size
Know how to interp[ret the graph:
Less than t value accept null hypothesis
Greater than t value accept a;lyterantaive hypothesis
*We will probably just do a paired t test.