lab exam - slides Flashcards
whats the reagent for lab 1
biuret
what does biuret do
biuret reaction for measuring protein concentration
Why BSA (what it stands for and 4 reasons)
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
describe the test tubes - increasing amounts of BSA
gets more purple
higher concentration of proteins
describe relationship between intensity, absorbance and transmission (lab 1)
I/I0 is proportional to % transmittance
I/I0 is 1/alpha (inversely proportional) to absorbance
transmission is 1/alpha to absorbance
what is the wavelength for absorbance by the biuret peptide colour
540 nm
explain the graph for lab 1
Absorbance = y
Concentrations mg/mL = x
goes through 0
linear trendline y=ax
linear proportional graph
Name the 3 separation techniques in lab 2
paper chromatography
cellulose acetate electrophoresis
SDS polyacrylamide gel electrophoresis
what is paper chromatography - lab 2
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
What is Rf - lab 2
Rf = a/b = moved/solvent front usually < 1
describe CAE - lab 2
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)
what is a pl - lab 2
pl = isoelectric point
pH at which net charge = 0
describe results of CAE - lab 2
Ponceau S
Lysozyme moves back towards neg pole
Albumin moves farthest towards pos pole
Casein doesn’t move
describe SDS PAGE - lab 2
Separation of proteins in a mixture by size - measured in KDa - 50KDa = 50 000g/mole
Proteins are denatured in process - primary structure left
Describe the denaturation in SDS PAGE - lab 2
BME - beta mercapto ethanol, SDS anionic detergent, BOILING (SDS coats proteins evenly with negative charge - BME disrupts disulfide bonds so tertiary and quaternary structures)
describe results of SDS PAGE - lab 2
Coomassie blue
Shows size of amino acids
moves furthest = smallest
describe amylase - lab 3
hydrolysis: starch +h20 -> maltose
fungal amylase
name the 3 conditions studied in lab 3 (affect amylase activity)
optimal pH
optimal temp
optimal enzyme concentration
describe amylase activity - lab 3
measured by rate of substrate (starch disappearing)
starch + iodine = blue
intensity of blue = concentration of starch
describe how blue is measured in lab 3
transmittance at 560 nm
describe transmission readings of lab 3
more starch = more blue = high absorbance SO LOWER TRANSMISSION
calibration of spectrophotometer = iodine so 100% transmittance
describe graph of lab 3
transmittance % = y
time = x
at end = no starch present - hydrolyzed
goes through origin
curves kinda flat at end
how to find rates for lab 3
go to 50% and make line down to x axis
0.5 x 50mg (initial) / time (ex: 10, 7, 12 mins…)
what is optimal condition for lab 3
when it peaks - highest y value
describe lab 4 - photosynthesis
4 pigments separated by paper chromatography
Solvent = petroleum ether (non polar) / acetone (polar)
name 4 pigments of lab 4 - from most to least polar
Chlorophyll b
Chlorophyll a
xanthophyll
beta carotene
its based on the amount of polar groups they have
what are the polar groups to count for pigments in lab 4
COH
CO
O
OCH3
Describe photosynthesis absorption VS action spectra
coincide
prove that chlorophylls are needed for photosynthesis
rate of photosynthesis (o2 release) = lowest around green wavelength - highest for chlorophyll a and b
state gen photosynthesis equation for lab 4
co2 + h20 ->(light!) sugar + o2
what is the iodine test - lab 4
Geranium leaves with out without - light and co2
do the leaves store starch?
state gen respiration formula - lab 4
glucose + o2 -> co2 + h20 + energy ATP
Describe respiration rate for lab 4
measured by o2 consumption with sensors
controlled experiment - dead peas = no o2 consumption, baseline, negative control, control for external factors - live peas = o2 consumption
describe respirometry graph o2 consumption by viable and dead pea - lab 4
dead peas = almost a line, just decreases a little
live peas = decreases a lot over time
graph doesnt start at origin
describe respirometry graph net o2 consumption per viable pea - lab 4
starts at origin
goes up - linear (ppm/time)
rate of cellular respiration = rate of o2 consumption per viable pea
ppm/min = slope
what is plasmid DNA - lab 5
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
describe gel filtration chromatography - lab 5
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
describe agarose gel electrophoresis - lab 5
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
what are restriction enzymes - lab 6
endonucleases
recognize and hydrolyze phosphodiesther linkage of both strands at palindromic site - same backwards and forwards
EcoRI
BamHI
HindIII
name and describe the two types of ends of restriction enzymes
blunt ends = middle split
sticky/flush ends - end split
Describe map of lambda DNA - lab 6
top = cutting sites
bottom = sizes of fragments
describe agarose gel electrophoresis in lab 6
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
describe graph of lab 6
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
describe specifics about fragment sizes in lab 6
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%
describe transformation of E COLI - lab 7 (3 elements + 3 plating mediums)
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
describe parts of pGLO DNA
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
describe ara operon regulation - lab 7
similar to lac operon
pBAD promoter
normally Ara operon codes for enzymes that breakdown arabinose
Green fluorescence with UV excitation
describe transformation results - 4 plates
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)
describe transformation efficiency - lab 7
number of transformants (colonies)/ug DNA
colonies / DNA ug x total vol/vol plated
total vol/vol plated >1
describe satelite colonies - lab 7
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
name/describe lac operon mutants - lab 7
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
what are lac operon mutants plated on - 4 things
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
describe bacterial lac operon - lab 7
repressed = glucose, no lactose
Induced = glycerol - NO glucose, IPTG - inducer ~ allolactose
describe IPTG and colour reaction
Conversion of X-Gal by B-galactosidase into blue indicator
