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My MCAT Cards > Lab Techniques > Flashcards

Lab Techniques Flashcards

1
Q

gel electrophoresis

A
  • 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
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2
Q

western blotting

A
  • 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
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3
Q

extraction

A

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

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4
Q

recrystallization

A

dissolve product in hot solvent
let it recrystallize as it cools
product must be soluble only at high temperatures

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5
Q

distillation
3 types

A

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&raquo_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

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6
Q

chromatography: general

A

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)

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7
Q

TLC

A

silica gel stationary, nonpolar solvent runs up
more NONpolar = higher it moves = higher Rf

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8
Q

column chromatography

A

column with polar silica
uses GRAVITY to move solvent + compound
polar sticks to column (slow), non-polar eludes FIRST

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9
Q

ion-exchange chromatography

A

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

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10
Q

size-exclusion chromatography

A

small pores that smaller molecules can fit into and travel in
so LARGER items elude FIRST

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11
Q

affinity chromatography

A

protein of interest bound to column with specific receptor/antibody
recover using eluent

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12
Q

gas chromatography

A

eluent is a GAS, travel through column with crushed metal/polymer at different rates

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13
Q

HPLC

A

high performance liquid chromatography
liquids under pressure
high performance due to computer control of process
otherwise similar to column/ion/affinity chormatography

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14
Q

southern vs northern blot

A
  • **southern (detect DNA)
  • northern (detect RNA)**
  • restriction enzymes, NaOH to ssDNA
  • after gel electrophoresis, radioactive-P labelled probe complementary to DNA of interest/RNA
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15
Q

polyacrylamide VS agarose

A

agarose = LARGER fragments of DNA/RNA
polyacrylamide = smaller

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16
Q

sanger sequencing

A
  • 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
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17
Q

chromatography (orgo)

A
  1. liquid chromatography (normal)
  2. HPLC
  3. gas chromatography
  4. size exclusion
  5. ion exchange
  6. affinity
  7. TLC
18
Q

distillation (orgo)

A
  1. simple
  2. fractional
  3. vacuum
19
Q

PCR

A
  1. denature = heated to 95 degrees, DNA separates
  2. annealing = cooled to allow primers to bind to ssDNA
  3. elongation = 72 degrees with Taq pol, new complementary strand made
20
Q

H-NMR (orgo)

A

order of shifts: alkane < alkene < halide < aromatic < aldehyde < COOH
splitting = n+1 (n = # of neighbouring hydrogens on adjacent C’s)

21
Q

IR spectroscopy (orgo)

A
  • only molecules with a dopole memoent show absorbance
  • x-axis is wavenumber, y axis is % absorbance
22
Q

UV-vis spectroscopy

A

of conjugated pi bonds increase, energy gap between HOMO and LUMO decreases (absorb higher wavelengths of light)

longer wavlength of light emitted

23
Q

autoradiography

A

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

24
Q

x ray crystallography

A

visualize structure of a molecule, often proteins
causes beam of x-ray to diffract in different directions
constructs 3D image by measuring angles

25
Q

immunoprecipitation

A

protein of interest precipitated from solution by adding bead-conjugated antibody

26
Q

mass spectrometry

A
  • 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
27
Q

enzyme linked immunoabsorbent assay (ELISA)

A
  • purpose = identify CONCENTRATION of a molecule of interest
  • use primary antibodies bound to secondary antibodies (visual)
28
Q

edman degredation

A
  • 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?)
29
Q

gram staining

A
  • 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
30
Q

restriction fragment length polymorphism

A

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

31
Q

restriction enzymes

A
  • recognize specifc palindromic sequence and cleave
32
Q

salting out

A
  • 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
33
Q

reducing sugar tests

A
  • 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
34
Q

tollen’s test

A
  • ketoses will not react so can distinguish between ketose and aldose
  • [Ag(NH3)2]NO3
35
Q

benedict’s reagent

A
  • ketose will react if alpha-hydroxy ketones (isomerize)
  • positive test = red precipitate formed
  • Cu2O (s) red
36
Q

fehling’s test

A
  • ketoses will react if alpha-hydroxy ketones (isomerize)
  • positive test = red precipitate (also Cu2O)
37
Q

cDNA libraries

A
  • 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
38
Q

role of boiling chips

A

provide a site for gas bubbles to form, preventing superheating

39
Q

role of vacuum distillation

A

to lower the boiling point by lowering pressure inside the flask

40
Q

sterilization

A
  • 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
41
Q

disinfection

A
  • disinfection refers to the removal of most microorganisms, especially pathogenic microorganisms
  • many disinfecting agents interfere with the cell walls of bacteria
  • eg. bleach
42
Q

cation vs anion exchange

A

cation exchange = separates positively charged proteins (column = negative)
anion exchange = separates negatively charged proteins (column = positive)

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