Lab Techniques Flashcards
gel electrophoresis
- separation of protein, DNA, RNA based on size + charge
- travel towards the + ANODE
- voltage source is applied, works like electrolytic cell
- native PAGE = non-denaturing
- SDS-PAGE = denatures with SDS (-), everything same charge, only mass seperates
- SDS only disrupts NON-covalent bonds
- reducing SDS-PAGE = disrupts covalent bonds too
- isoelectric focusing = separates proteins based on pI
western blotting
- purpose = detect presence of SPECIFIC PROTEIN
- performed after SDS-PAGE separation
- add fluorescently labelled antibody specific to protein (or 2 antibodies)
- if detect antibody presence means protein is there
extraction
use 2 layers that don’t mix (aqueous + organic)
use separatory funnel to drain 1st
H bonds likely in aqueous, dipole-dipole in organic, etc.
after solvent gathered, evaporate to get product
recrystallization
dissolve product in hot solvent
let it recrystallize as it cools
product must be soluble only at high temperatures
distillation
3 types
separates 2 liquids based on differences in boiling point
1. simple distillation = BP < 150 degrees C, at least 25 degrees apart
2. vaccuum distillation = BP»_space; 150 degrees – vacuum decreases ambient pressure, less temp needed to increase vapour pressure base boiling point, prevents degredation
3. fractional distillation = BP close together, use longer column with high SA to allow distillate more places to condense back down, REFINED SEPARATION
chromatography: general
more similar a compound to its surroundings, more it sticks and SLOWER it moves
Rf = distance spot moved / distance solvent moved (higher = moved further = more similar to MOBILE phase not stationary)
TLC
silica gel stationary, nonpolar solvent runs up
more NONpolar = higher it moves = higher Rf
column chromatography
column with polar silica
uses GRAVITY to move solvent + compound
polar sticks to column (slow), non-polar eludes FIRST
ion-exchange chromatography
beads in column coated with ions
SLOWEST will be products with opposite charge to ions in column (sticky)
use a SALT at the end to elude the compound of interest stuck
size-exclusion chromatography
small pores that smaller molecules can fit into and travel in
so LARGER items elude FIRST
affinity chromatography
protein of interest bound to column with specific receptor/antibody
recover using eluent
gas chromatography
eluent is a GAS, travel through column with crushed metal/polymer at different rates
HPLC
high performance liquid chromatography
liquids under pressure
high performance due to computer control of process
otherwise similar to column/ion/affinity chormatography
southern vs northern blot
- **southern (detect DNA)
- northern (detect RNA)**
- restriction enzymes, NaOH to ssDNA
- after gel electrophoresis, radioactive-P labelled probe complementary to DNA of interest/RNA
polyacrylamide VS agarose
agarose = LARGER fragments of DNA/RNA
polyacrylamide = smaller
sanger sequencing
- determine DNA sequence using ddNTPs without 3’OH
- gel – smallest fragments (5’) tavel to bottom of gel (this is 3’ of original strand)
- read gel bottom - top (5’ - 3’) then synthesize complementary for original
chromatography (orgo)
- liquid chromatography (normal)
- HPLC
- gas chromatography
- size exclusion
- ion exchange
- affinity
- TLC
distillation (orgo)
- simple
- fractional
- vacuum
PCR
- denature = heated to 95 degrees, DNA separates
- annealing = cooled to allow primers to bind to ssDNA
- elongation = 72 degrees with Taq pol, new complementary strand made
H-NMR (orgo)
order of shifts: alkane < alkene < halide < aromatic < aldehyde < COOH
splitting = n+1 (n = # of neighbouring hydrogens on adjacent C’s)
IR spectroscopy (orgo)
- only molecules with a dopole memoent show absorbance
- x-axis is wavenumber, y axis is % absorbance
UV-vis spectroscopy
of conjugated pi bonds increase, energy gap between HOMO and LUMO decreases (absorb higher wavelengths of light)
longer wavlength of light emitted
autoradiography
imaging technique part of souther/northern/western blot
radiation of labelled substance placed by photographic emulsion with silver halide
radiation converts silver halide to metallic silver
x ray crystallography
visualize structure of a molecule, often proteins
causes beam of x-ray to diffract in different directions
constructs 3D image by measuring angles
immunoprecipitation
protein of interest precipitated from solution by adding bead-conjugated antibody
mass spectrometry
- to determine the molecular weight of a compound and determine molecular structure
- sample vaporized and ionized, charged molecule collides with electron causing ejection of an electron (radical) – radicals can be detected
enzyme linked immunoabsorbent assay (ELISA)
- purpose = identify CONCENTRATION of a molecule of interest
- use primary antibodies bound to secondary antibodies (visual)
edman degredation
- purpose = sequence AA’s in a protein, from N-terminal
- creates PTH-AA hybrid (n-term) that breaks off and can be identified with HPLC (it is coloured?)
gram staining
- stain bacteria with crystal violet (purple) use safranin as counter stain (pink)
- gram positive = purple – thick peptidoglycan takes up stain
- gram negative = pink – think peptidoglycan, LPS, takes up safranin
restriction fragment length polymorphism
to identify diffrences in domologous DNA sequences based on lengths of fragments caused by retriction enzymes
usually didfferences in 2+ DNA sequence in the SAME gene
restriction enzymes
- recognize specifc palindromic sequence and cleave
salting out
- purpose = to purify proteins in a sample
- adding salts can selectively precipitate out proteins due to competition between salt ions and protein for water molecules
reducing sugar tests
- purpose = identify reducing sugar in solution
- reducing sugar = has free OH group on anomeric carbon that can be OXIDIZED (act as reducing agent)
- so hemiacetals are reducing, acetals are NOT
- note: ketoses are reducing due to isomaerization to aldehyde form
tollen’s test
- ketoses will not react so can distinguish between ketose and aldose
- [Ag(NH3)2]NO3
benedict’s reagent
- ketose will react if alpha-hydroxy ketones (isomerize)
- positive test = red precipitate formed
- Cu2O (s) red
fehling’s test
- ketoses will react if alpha-hydroxy ketones (isomerize)
- positive test = red precipitate (also Cu2O)
cDNA libraries
- purpose = create cDNA stands that can be expressed in bacterial vectors
- can’t insert eukaryotic DNA directly in, due to presence of INTRONS
- instead, make cDNA from complementary mRNA (no introns) with reverse transcriptase
role of boiling chips
provide a site for gas bubbles to form, preventing superheating
role of vacuum distillation
to lower the boiling point by lowering pressure inside the flask
sterilization
- Sterilization refers to completely removing all living microorganisms and viruses from the surface of an inanimate object
- autoclave = subjecting a surface to saturated steam under high pressure at a temperature well over the boiling point of water. When correctly done, autoclaving will inactivate all bacteria, spores, fungi, and viruses
- autoclave = MOST EFFECTIVE
- other examples: UV, heat, chemicals
disinfection
- disinfection refers to the removal of most microorganisms, especially pathogenic microorganisms
- many disinfecting agents interfere with the cell walls of bacteria
- eg. bleach
cation vs anion exchange
cation exchange = separates positively charged proteins (column = negative)
anion exchange = separates negatively charged proteins (column = positive)
