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bio 2 > lab exam - slides > Flashcards

lab exam - slides Flashcards

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Biology 101 flashcards
1
Q

whats the reagent for lab 1

A

biuret

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2
Q

what does biuret do

A

biuret reaction for measuring protein concentration

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3
Q

Why BSA (what it stands for and 4 reasons)

A

Bovine serum albumin

readily available
low cost of production
it produces a signal in protein assays that increases linearly with increasing protein
average sized protein

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4
Q

describe the test tubes - increasing amounts of BSA

A

gets more purple
higher concentration of proteins

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5
Q

describe relationship between intensity, absorbance and transmission (lab 1)

A

I/I0 is proportional to % transmittance
I/I0 is 1/alpha (inversely proportional) to absorbance
transmission is 1/alpha to absorbance

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6
Q

what is the wavelength for absorbance by the biuret peptide colour

A

540 nm

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7
Q

explain the graph for lab 1

A

Absorbance = y
Concentrations mg/mL = x
goes through 0
linear trendline y=ax
linear proportional graph

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8
Q

Name the 3 separation techniques in lab 2

A

paper chromatography
cellulose acetate electrophoresis
SDS polyacrylamide gel electrophoresis

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9
Q

what is paper chromatography - lab 2

A

Separates amino acids based on their polarity and their solubility in a given solvent
stationary phase and mobile phase
most polar moves the least
most hydrophobic (least polar) moves the most

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10
Q

What is Rf - lab 2

A

Rf = a/b = moved/solvent front usually < 1

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11
Q

describe CAE - lab 2

A

Separation of proteins by charge (most proteins have charges bc of side chains, net charge is affected by surrounding pH of solution)
Proteins are partially denatured in process ( at pH 8.8, ionic interactions are affected compared to physiological pH, BUT covalent bonds and disulfide bonds are not affected)

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12
Q

what is a pl - lab 2

A

pl = isoelectric point
pH at which net charge = 0

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13
Q

describe results of CAE - lab 2

A

Ponceau S
Lysozyme moves back towards neg pole
Albumin moves farthest towards pos pole
Casein doesn’t move

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14
Q

describe SDS PAGE - lab 2

A

Separation of proteins in a mixture by size - measured in KDa - 50KDa = 50 000g/mole
Proteins are denatured in process - primary structure left

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15
Q

Describe the denaturation in SDS PAGE - lab 2

A

BME - beta mercapto ethanol, SDS anionic detergent, BOILING (SDS coats proteins evenly with negative charge - BME disrupts disulfide bonds so tertiary and quaternary structures)

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16
Q

describe results of SDS PAGE - lab 2

A

Coomassie blue
Shows size of amino acids
moves furthest = smallest

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17
Q

describe amylase - lab 3

A

hydrolysis: starch +h20 -> maltose
fungal amylase

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18
Q

name the 3 conditions studied in lab 3 (affect amylase activity)

A

optimal pH
optimal temp
optimal enzyme concentration

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19
Q

describe amylase activity - lab 3

A

measured by rate of substrate (starch disappearing)
starch + iodine = blue
intensity of blue = concentration of starch

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20
Q

describe how blue is measured in lab 3

A

transmittance at 560 nm

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21
Q

describe transmission readings of lab 3

A

more starch = more blue = high absorbance SO LOWER TRANSMISSION
calibration of spectrophotometer = iodine so 100% transmittance

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22
Q

describe graph of lab 3

A

transmittance % = y
time = x
at end = no starch present - hydrolyzed
goes through origin
curves kinda flat at end

23
Q

how to find rates for lab 3

A

go to 50% and make line down to x axis
0.5 x 50mg (initial) / time (ex: 10, 7, 12 mins…)

24
Q

what is optimal condition for lab 3

A

when it peaks - highest y value

25
Q

describe lab 4 - photosynthesis

A

4 pigments separated by paper chromatography
Solvent = petroleum ether (non polar) / acetone (polar)

26
Q

name 4 pigments of lab 4 - from most to least polar

A

Chlorophyll b
Chlorophyll a
xanthophyll
beta carotene

its based on the amount of polar groups they have

27
Q

what are the polar groups to count for pigments in lab 4

A

COH
CO
O
OCH3

28
Q

Describe photosynthesis absorption VS action spectra

A

coincide
prove that chlorophylls are needed for photosynthesis
rate of photosynthesis (o2 release) = lowest around green wavelength - highest for chlorophyll a and b

29
Q

state gen photosynthesis equation for lab 4

A

co2 + h20 ->(light!) sugar + o2

30
Q

what is the iodine test - lab 4

A

Geranium leaves with out without - light and co2
do the leaves store starch?

31
Q

state gen respiration formula - lab 4

A

glucose + o2 -> co2 + h20 + energy ATP

32
Q

Describe respiration rate for lab 4

A

measured by o2 consumption with sensors
controlled experiment - dead peas = no o2 consumption, baseline, negative control, control for external factors - live peas = o2 consumption

33
Q

describe respirometry graph o2 consumption by viable and dead pea - lab 4

A

dead peas = almost a line, just decreases a little
live peas = decreases a lot over time
graph doesnt start at origin

34
Q

describe respirometry graph net o2 consumption per viable pea - lab 4

A

starts at origin
goes up - linear (ppm/time)

rate of cellular respiration = rate of o2 consumption per viable pea
ppm/min = slope

35
Q

what is plasmid DNA - lab 5

A

small circular double stranded DNA - contains important but not essential info
ROLES = in bacteria - transfer and acquisition of genes like antibiotic resistant genes, for biologists = recombinant DNA technologies

Plasmid DNA is extracted from bacteria by miniprep (maxiprep) procedure - bacterial RNA contaminates plasmid DNA preparations

36
Q

describe gel filtration chromatography - lab 5

A

cellulose porous beads - holes and channels
gravity driven
separates molecules by SIZE AND SHAPE
plasmid DNA = large and bulky - gets out first
bacterial RNA = Small and compact = enters holes and is trapped longer
initially = void vol, between beads, then DNA then DNA and RNA and lastly just RNA

37
Q

describe agarose gel electrophoresis - lab 5

A

agarose gel matrix with microscopic pores - molecular sieve
uses electric field
Separates by SIZE AND SHAPE
large molecules - DNA = hard to move across gel - PLASMID DNA HIGHER UP
small molecules - move faster - migrates to positive pole - RNA LOWER
methylene blue STAIN

38
Q

what are restriction enzymes - lab 6

A

endonucleases
recognize and hydrolyze phosphodiesther linkage of both strands at palindromic site - same backwards and forwards

EcoRI
BamHI
HindIII

39
Q

name and describe the two types of ends of restriction enzymes

A

blunt ends = middle split
sticky/flush ends - end split

40
Q

Describe map of lambda DNA - lab 6

A

top = cutting sites
bottom = sizes of fragments

41
Q

describe agarose gel electrophoresis in lab 6

A

uncut DNA shouldnt move at all
then the cut one should move - measure with ruler to get distance migrated and we are given one set of fragment sizes (BP) - HindIII

42
Q

describe graph of lab 6

A

Fragment size bp = y - LOG SCALE
distance migrated in cm = x
y=kx^p = negative curve down, power function = trendline
use this to estimate sizes of BamHI and EcoRI - measure dist migrated and estimate using graph/function

43
Q

describe specifics about fragment sizes in lab 6

A

Large fragments - 10 000bp or medium - 5000 bp and if close in size - do not resolve by agarose gel electrophoresis - APPEAR as one band
—> lower agarose gel % to get better resolution -
Small fragments - less than 5000bp run out of gel - increase agarose gel%

44
Q

describe transformation of E COLI - lab 7 (3 elements + 3 plating mediums)

A

competency - treatment with CaCl2
mixing with DNA (t) - without DNA - control untransformed
transformation - heat shock
plating on LB medium - without ampicillin, with ampicillin - selective medium, with ampicillin and arabinose - selective inducing medium

45
Q

describe parts of pGLO DNA

A

ori - origin of replication
araC - gene for araC bifunctional regulatory protein - repressor protein
Pbad = arabinose promoter
GFP = green fluorescent protein gene under control of Pbad
bla - b-lactamase gene - ampicillin resistance

46
Q

describe ara operon regulation - lab 7

A

similar to lac operon
pBAD promoter
normally Ara operon codes for enzymes that breakdown arabinose
Green fluorescence with UV excitation

47
Q

describe transformation results - 4 plates

A

Untransformed -pGLO plasmid - control = LB - cell growth and LB/amp - no cell growth, quality of ampicillin
transformed +pGLO plasmid = LB/Amp -AMP^R colonies, no GFP production and LB/Amp/Ara - AMP^R colonies, arabinose induced GFP production (glows)

48
Q

describe transformation efficiency - lab 7

A

number of transformants (colonies)/ug DNA

colonies / DNA ug x total vol/vol plated

total vol/vol plated >1

49
Q

describe satelite colonies - lab 7

A

untransformed
AmpS - smaller, appear later after transformed bacteria have produced B-lactamase and inactivated ampicillin around - NO GFP
grow in antibiotic resistant area but arent antibiotic resistant

50
Q

name/describe lac operon mutants - lab 7

A

WT = wild type lac operon - no mutations
lac z- = defective lacZ gene - no beta-galactosidase produced
lac Oc = constitutive lac operon - mutation repressor doesnt bind - always on

51
Q

what are lac operon mutants plated on - 4 things

A

McConkey - lactose metabolism
LB glycerol with IPTG - lac operon inducing conditions
LB glucose - lac operon repressing conditions
X-gal indicator measures lac operon activity on LB plates

52
Q

describe bacterial lac operon - lab 7

A

repressed = glucose, no lactose
Induced = glycerol - NO glucose, IPTG - inducer ~ allolactose

53
Q

describe IPTG and colour reaction

A

Conversion of X-Gal by B-galactosidase into blue indicator

bio 2 (6 decks)

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