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Lab test - Coggle Diagram
Lab test
Lab 3
Separation techniques
paper chromatography
Separation of amino acid
Based on polarity and solubility
Non polar/hydrophobic moves the most
Solvent 2 phases :
stationary phase--> formic acid
mobile phase--> isopropanol
Ninhydrin needed
cellulose acetate electrophoresis
separation of proteins
based on charge
Proteins are partially denatured in the process
carried out at pH 8.8
Proteins are stained with
Ponceau-S
Results
Lysozyme--> most positive
Albumin--> most negative
SDS polyacrylamide
Separation of proteins
separation by size
proteins are denatured in the process
proteins are stained with
coomassie blue
SDS--> gives negative charge
Lab 1
Protein standard curve and identification of unknown
Reagent
Biuret
Becomes darker purple when it comes in contact with proteins
540 nm
Bovine serum albumin
5 mg/ml
Spectrophotometer
For calculations
Find slope and plug in absorbance (y) to find concentration (x)
Lab 2
Restriction digestion of Lambda DNA
Blunt ends vs sticky ends
Sticky ends for recombinant DNA
3 enzymes
BamHI EcoRI HindIII
HindIII travels the furthest
BamHI --> 5
EcoRI --> 5
HindIII --> 7
Number of segments -->
Agarose gel electrophoresis
how far the segments of DNA travel based on their size
Dye that binds to the DNA molecules
Methylene blue
How is the DNA of the host protected
Because the bases are methylated at the sites recognized by endonuclease
Lab 4
Amylase activity as a function of pH and enzyme concentration
Starch + iodine--> dark blue
iodine alone=100% transmission
calibration of spectrophotometer
more starch
more blue
higher absorbance
lower transmission
560 nm
optimal concentration (0,03 mg/ml) and pH (5) conditions
1) amylase cleaves starch into smaller subunits--> maltose
2) Enzyme maltase converts maltose to glucose
3) can further be broken down into carbon dioxide, water and energy
Hydrolysis of starch
substrate -->starch
Lab 5
Metabolic pathways
Factors in photosynthesis
Iodine test:
Presence of light and Co2
Dark purple-photosynthesis occured
Chromatography
2 possible solvents : ether--> non polar acetone--> polar
from non polar to polar:
beta carotene
xanthophyll
chlorophyll a
chlorophyll b
Tetrazolium test
testing the viability
if they turn pink --> positive
with tetrazolium solution
Respirometry
mesuring oxygen consumption
if peas are alive
consume oxygen
if peas are dead
do not consume oxygen
Lab 7
Lac operon mutants
bacterial cells must be competent
how?
treated with CaCl2
transformation : Heat shock
plating on LB medium
with ampicillin
With ampicillin and arabinose
without ampicillin
pGLO
araC
PBad
GFP
bla--> b-lactamase gene- ampicillin resistance
ori
satellite colonies
untransformed
transformed bacteria produce b-lactamase and inactivate ampicillin around
no GFP production --> do not glow
Lac operon demo :
3 strains
Lac Z+
Lac Z-
Lac Oc
plated on :
Mc Conkey
lactose metabolism
X-gal plates
LB glycerol with IPTG
inducing conditions
LB glucose
repressing conditions
Lab 6
Gel filtration chromatography and agarose gel electrophoresis
1) Gel filtration chromatography
size and shape
cellulose porous beads--> matrix
elution buffer --> mobile phase (flows through matrix)
total volume of buffer between beads--> void volume
Bed volume --> beads + void volume
Result :
Bacterial RNA gets stuck in beads because its smaller
Plasmid DNA comes out first because its larger and heavier
2) Agarose Gel electrophoresis
matrix with microscopic pores
size and shape
methylene blue
Result :
DNA is heavier so it doesnt travel as far
supercoiled DNA travels a bit further
Bacterial RNA is lighter so it travels further
Separation of nucleic acids