Lab Techniques

Question 1

gel electrophoresis

Answer 1

• 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

Question 2

western blotting

Answer 2

• 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

Question 3

extraction

Answer 3

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

Question 4

recrystallization

Answer 4

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

Question 5

distillation
3 types

Answer 5

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

Question 6

chromatography: general

Answer 6

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)

Question 7

TLC

Answer 7

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

Question 8

column chromatography

Answer 8

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

Question 9

ion-exchange chromatography

Answer 9

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

Question 10

size-exclusion chromatography

Answer 10

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

Question 11

affinity chromatography

Answer 11

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

Question 12

gas chromatography

Answer 12

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

Question 13

HPLC

Answer 13

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

Question 14

southern vs northern blot

Answer 14

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

Question 15

polyacrylamide VS agarose

Answer 15

agarose = LARGER fragments of DNA/RNA
polyacrylamide = smaller

Question 16

sanger sequencing

Answer 16

• 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

Question 17

chromatography (orgo)

Answer 17

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

Question 18

distillation (orgo)

Answer 18

1. simple
2. fractional
3. vacuum

Question 19

PCR

Answer 19

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

Question 20

H-NMR (orgo)

Answer 20

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

Question 21

IR spectroscopy (orgo)

Answer 21

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

Question 22

UV-vis spectroscopy

Answer 22

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

longer wavlength of light emitted

Question 23

autoradiography

Answer 23

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

Question 24

x ray crystallography

Answer 24

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

Question 25

immunoprecipitation

Answer 25

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

Question 26

mass spectrometry

Answer 26

* 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

Question 27

enzyme linked immunoabsorbent assay (ELISA)

Answer 27

* purpose = identify CONCENTRATION of a molecule of interest * use primary antibodies bound to secondary antibodies (visual)

Question 28

edman degredation

Answer 28

* 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?)

Question 29

gram staining

Answer 29

* 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

Question 30

restriction fragment length polymorphism

Answer 30

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*

Question 31

restriction enzymes

Answer 31

* recognize specifc palindromic sequence and cleave

Question 32

salting out

Answer 32

* 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

Question 33

reducing sugar tests

Answer 33

* 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**

Question 34

tollen's test

Answer 34

* **ketoses will not react** so can distinguish between ketose and aldose * [Ag(NH3)2]NO3

Question 35

benedict's reagent

Answer 35

* ketose will react if alpha-hydroxy ketones (isomerize) * positive test = red precipitate formed * Cu2O (s) red

Question 36

fehling's test

Answer 36

* ketoses will react if alpha-hydroxy ketones (isomerize) * positive test = red precipitate (also Cu2O)

Question 37

cDNA libraries

Answer 37

* 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

Question 38

role of boiling chips

Answer 38

provide a site for gas bubbles to form, preventing superheating

Question 39

role of vacuum distillation

Answer 39

to lower the boiling point by lowering pressure inside the flask

Question 40

sterilization

Answer 40

* 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

Question 41

disinfection

Answer 41

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

Question 42

cation vs anion exchange

Answer 42

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