Gen bio 2 lab fianl Flashcards
(34 cards)
1
Q
Turnip Enzyme
A
10 g white part of turnip was blended mixed with water an then filtered. filteredd then combined with 200-400 mL of diH2O depending on how old the turnip is.
Cold temperature used to keep enzymes from denaturing
2
Q
Assay procedure
A
- Blank assay is to calibrate the colorimeter
- Blank has everything minus colored parts
- Cuvette, guaiacol, water, hydrogen peroxide - Measurement set at 500 nm
- Mix everything, then add the enzyme
- measured transmittance from 70%-50%
3
Q
Effect of Substrate concentration on reaction rate
A
- greater substrate creates greater reaction rate until saturation point (H2O2)
- Peroxidase requires guaiacol and h2o2
- C1V1 = C2V2, .1% conc H2O2 at start (assay 3) and 1.6 mL, to 5 mL vol, C2 = .032
4
Q
Effect of enzyme concentration on reaction rate
A
- Assay 13 had the highest enzyme conc, so therefor it should have the smallest reaction time (fastest)
5
Q
Milk Protein and Centrifuge
A
- Acid and Organic solvent denature proteins
- When the proteins are dissolved and become suspended in solution
- Centrifuge can now separate it into pellets
- Two 28 mL milk solutions, tube A had higher protein concentrations, so bigger pellet
6
Q
Effect of pH on enzyme activity
A
- High pH more protein denaturing, decreasing reaction rate
- Adding base after acid does not ‘fix’ enzymes
7
Q
Calculating pH
A
- To calculate the pH of each assay tube, use C1V1 = C2V2
- Solve for C2 which is also the [H+].
- Plug [H+] in the equation pH = -log [H+]. For example, to get assay 2’s pH, the C1 = 2.5 X 10^-7 M, V1 = 2mL, and V2 = 5mL
C2 = 1 * 10^-7
8
Q
Effect of temperature on enzyme activity
A
- Higher temperature means more denaturation
9
Q
Effects of organic solvent on enzyme activity
A
- Organic solvent has alot of water and other solvent in it which tends to denature proteins
10
Q
Effect of irreversible metabolic inhibitor
A
- Fluoride acts as an inhibitor for this specific reaction, and bring the reaction rate to zero
- doubling the substrate does not increase reaction rate, letting us know the fluoride is not a competitive inhibitor
11
Q
Effect of reversible inhibitor
A
- Reversible means it is competitive
- So by increasing the substrates we can increase the reaction rate
12
Q
What is t-test used for?
A
- is there any statistical difference between two populations and their means
13
Q
What are the variables in a t-test?
A
- xbar 1 (mean 1)
- xbar 2 (mean 2)
- N 1 (population size 1)
- N 2 (population size 2)
- S1 ( stdev 1)
- s2 ( stdev 2)
- df ( n1+n2-2)
- use .2 probability
14
Q
T-test calculation
A
After getting your t-value, if your calculated T is less than the critical T you fail to reject Ho
15
Q
Accuracy vs. Precision
A
- Accuracy is when you are close to the real truth
- Precision is when you are consistent
16
Q
Relating mass
A
- allows you to estimate the area of an irregular shape
- find mass of an object (paper) and its area
- cut this object (paper) into an irregular shape
- use mass as a conversion factor for area
- mass (uncut)/ area (uncut) = mass (irregular)/area (irregular)
17
Q
What is turbidimetry?
A
- Using colorimeter to measure how much light is reflected by suspended particles in a solution
- more scattering means less % transmitance
18
Q
Turbidimetry Example:
A
- dissolved .5 g yeast into 25mL GYE solution
- yeast conc is 5 X 10^6 cells per mL
- 1/10 dilution is 1 mL stock with 9 mL GYE tube 1 (1:10)
- then a 3/10 dilution from first dilution, 3 mL from tube 1 and 7 mL stock (3:100)
- to get conc of yeast, take original conc and divide it by dilution factor
- higher conc more scattering of light, less transmittance
19
Q
Chromatography
A
- used to separate components of a complex mixture
- We did paper chromatography, mix of 3 types of dye at one point
- With water creeping up to point, separates three dyes by their affinity for paper/water
20
Q
Bacillus subtilis
A
- gram-positive
- motile
21
Q
Escherichia coli
A
- gram negative
- bacillus
- motile
22
Q
Micrococcus luteus
A
- gram-positive
- non motile
- coccus
- yellow pigment
23
Q
Serratia marcescens
A
- Gram negative
- motile bacillus
- red pigment
24
Q
Positive stains
A
- all bind to a specific cellular component
- methylene blue is a positive stain used
- stains DNA
- oil immersion is 100x, use with immersion oil for proper functioning
25
Negative Stains
- stains background, not cell
- used nigrosin
26
Safranin Stain
- positive
- used to show size difference between e. coli and b. subtilis
- can stain both gram positive and negative species
- crystal violet can only stain gram positive
27
Metabolic Test
- tested for the enzyme catalase (breaks down h2o2)
- M. Luteus released bubbles when in contact with h2o2, it has catalase
28
The Gram Stain
- B. subtilis (g-pos) and E.coli (g-neg) compared
- g-pos have thicker peptidoglycan wall
- crystal violet used first to stain g-pos, doesnt stain g-neg
- Safranin then used, g-pos remain violet, safranin can't stain them anymore and it stains g-neg
29
Bacterial Capsule
- negative red stain used to dye background
- then red stain used to dye the cell
- left with red background, white capsule, red cell
30
Chi Squared
- Sum of (observed - expected)^2/expected
- if calculated chi sq > chi sq critical reject the null
31
How does peroxidase work?
- uses H2O2 to oxidize a phenolic compound (guaiacol) to reduce h2o2 to h2o, make quinone and methanol
32
What is the main protein in milk?
Casein
33
Moving vs. Stationary phase in chromatography
- Stationary refers to the moment pre-solvent, when the solute is on the paper
- Moving refers to when the solvent is added to the paper
- Some molecules have a higher affinity for the stationary phase (not moved by solvent) while others have a higher affinity for the moving phase (when solvent takes it)
34
rf
distance traveled by solute / distance traveled by solvent
