Final Exam Review

BCHS 3201
Jessica Stevens
Teaching Assistant

Fall 2022
 Lab Math
• Making solutions from stock c1V1=c2V2
• % solutions, w/v (1g  1mL) and v/v
• X dilutions (1X, 2X etc)
• Molarity:
 Pipetting

Pipettor Theoretical Recommended

Range (l) Range (l)
P10 0-10 1-10
P20 0-20 2-20
P200 0-200 20-200
P1000 0-1000 100-1000
 Plasmid Purification – Reaction Components
Solution 1:
• What is the purpose of EDTA?
– Chelate divalent metal cations so
they are not available to DNAses as
cofactors. Otherwise, they would
destroy the plasmid DNA
• Glucose
– Prevent premature cell lysis by
osmotic shock
• Alkaline pH (Tris pH=8)
reduces electrostatic interactions
between DNA and scaffolding proteins
Solution 2, lysis buffer:
• SDS
– Disrupt cell membrane (amphipatic
detergent, interacts with and disrupts
amphiphatic phospholipid bilayer of
membrane
• NaOH
– Denatures chromosomal & plasmid DNA
Solution 3:
• Sodium Acetate (pH=5.5)
– Neutralize the lysis buffer
– Basepairing of the plasmid DNA is restored
– Precipitates the
proteins/lipids/chromosomal DNA out
 Plasmid Purification
• Why does plasmid DNA renature fully,
but chromosomal does not?
– Topologically intertwined, smaller
– RNA will also stay in solution
• Why 90% Ethanol?
– Precipitates DNA: it’s less polar than
water and allows phosphate backbone
to interact with sodium ions.
• Why 70% Ethanol?
– For washing: enough to prevent DNA
from going back in solution, but allow
washing off excess salt
• Why TE/RNAse
– DNA more stable in TE buffer
– RNAse to remove soluable RNA and
avoid smears in gel
• Agarose Gel
– Loading buffer: tracking dye, glycerol:
higher density, makes DNA stay in
bottom of the wells
– DNA is negatively charged (in pH=7),
migrates towards positive pole
– Separation by size, charge and shape
– The higher the agarose %, the higher
the separation power (resolution)
 Agarose Gel Electrophoresis Marker

• Which DNA fragments

Linear
run faster?
– Smaller Nicked, circular

Supercoiled, native
• How does Ethidium condensed form
runs fastest
Bromide/GelGreen work?
– Fluorescent dye, visible in Plasmid size: 2686 bp
UV, intercalates between
base pairs

• Why is GelGreen safer

the EtBr?
– Cannot pass through cell
membrane
RNA
 Restriction Enzymes
• Cleaves terminal residues from linear DNA
– Exonuclease
• Cleaves internal residues
– Endonuclease
• Recognizes the same sequence & cleaves at the same site
– Isoschizomer
• Recognizes the same sequence & cleaves at different sites
– Neoschizomer
• Off-site cleavage due to non-standard conditions
– Star activity
 Restriction Digest
0 • Enzyme 1
– Cleaves at 500bp site

• Enzyme 2
– Cleaves at 1000bp site
Plasmid X
2,000 bp
500 • Digest with Enzyme 1
– Fragment #
– Fragment size(s)

• Digest with Enzyme 1 + Enzyme 2

– Fragment #
1,000 – Fragment size(s)
– Incomplete digest fragment #
 Polyacrylamide gels
• Used when resolving proteins or
DNA fragments of nearly the same
size (in your case serum Proteins!)
• Made out of crosslinked
polymerized Acrylamide and bis-
Acrylamide
• Native (size, shape, charge, size
cannot be determined) vs. SDS PAGE
(deantured, equally charged, only
based on M.W.)
• Stacking vs. Resolving gel
• Coomassie Brilliant Blue: non-
specifically stains all proteins
• Just the M.W. does not proof the
purified protein’s identity
• Western Blot: Proteins from gel are
electrophoretically transferred to
membrane, membrane is blocked
to prevent unspecific binding,
protein of interest is identified
through antibody binding coupled
to colorimetric (etc.) labeling
 Polyacrylamide Gel Electrophoresis
• Purpose of SDS
– Coat proteins with uniform negative charge, disrupt native structure
• Migrate based on size, not charge

• Purpose of ß-Mercaptoethanol
-Reduction (“break”) of disulfide bridges

• Coomassie vs Western Blot

– Which is more specific? Why?
• Western blot – antibodies directed to a specific protein of interest
 *
* * **
Classic Glass slide Microarray *
Affymetrix type Gene chip
To compare gene expression, RNA is extracted 5µm
from sample and transcribed to cDNA.
5µm
Millions of
identical
probes / feature

5”
1.28cm
Up to
~6,500,000
features / chip

1.28cm Affymetrix Educator Resources

• Chips are synthesized onto silicate glass surface using

photolithographic synthesis to create different probes for each
feature simultaneously
• Each feature contains probes specific to one gene of the organism
• cDNA is transcribed to Biotin labeled RNA -> visualized with Biotin-
binding Fluorophore
• Two compare 2 samples, each will be added to a separate chip and
http://www.vetmed.iastate.edu/faculty_staff/users/phillips/Micro402/17-genomics/microarray.htm intensity of fluorescence is compared for each feature
 X-Ray Crystallography
• What is diffraction data • Max Von Laue
converted into? – Showed X-rays were a form of
– Electron density map electromagnetic radiation
– Crystals are periodic arrangement
of atoms
• Bragg’s Law
– Angle of light scattering
• Role of PEG
– William Henry & William Lawrence
Bragg – Crowding agent
• Hanging drop method
Ionic strength of the buffer in the droplet • Role of NaCl
is higher or lower than the reservoir? – Keeps the protein soluble
-lower!
 Bonus Question

• A method to solve protein structures requiring a beam of x-rays

resulting in multiple solutions to a set of equations was
developed by…?
– The Braggs!!
 Size Exclusion Chromatography
• Stokes radius
– Radius of a molecule as it tumbles
through a column

• Smaller or larger molecules will

elute first in S.E.C.? Why?
– Larger; will not penetrate the gel
beads’ pores as far as smaller
molecule.
 Kinetics
k1
E  S  ES 
k2
E  P
k -1

Peroxidase
 Kinetics
k1
E  S  ES 
k 22
E  P
k -1

Vmax S
vo 
K M  S

The Michaelis - Menten equation

v0 : Initial velocity (linear incline of reaction speed)
measured for multiple Substrate concentrations.
The Km is the substrate concentration where vo equals one-
half Vmax
 The Double Reciprocal Plot or
Lineweaver-Burk Plot

1  KM  1 1
  
vo  Vmax  S Vmax

Compare to y= mx+ b
There are three types of
inhibition kinetics:

Competitive

Mixed

Uncompetitive