(M) L2.1 : Gel Electrophoresis Flashcards
commonly used to separate DNA fragments from each other
electrophoresis
we use electrophoresis to see the presence and sizes of ______
DNA fragments
can be used to analyze restriction-digestion of products
electrophoresis
Electrophoresis is a method of separating __________ _________ substances in a mixture
electrically charged substances
enumerate the three electrically charged substances mentioned
DNA, RNA, and proteins
where is the sample of the mixture is placed on?
supporting medium
use this card to familiarize examples of supporting medium (or enumerate if you’re a masochist)
paper
starch gel
cellulose acetate
agarose gel
polyacrylamide gel
Selecting the type of supporting medium depends on the _____
experiment
a technique used for separation of charged molecules such as DNA, RNA, or protein molecules through liquid or gel medium according to their size and charge using an electrical current
electrophoresis
negative or positive electrode?
net positive charge migrates toward the cathode?
negative electrode
negative or positive electrode?
net negative charge move toward the anode
positive electrode
this allows the biomolecules to migrate towards the electrode of opposite charge
electrostatic attraction
the ________ in electrical field depends on its net charge, size, shape, strength of electrical field, properties of supporting medium and temperature
rate of migration of an ion
factors that affect the rate of migration of an ion
net charge
size
shape
strength of electrical field
these molecules have positive or negative charges
biological molecules
what is the charge of biological molecules in acidic conditions?
positively charged
what is the charge of biological molecules in alkaline conditions?
negatively charged
who pioneered the work on moving boundary electrophoresis in 1930?
Arne Tiselius
What did Arne Tiselius developed after moving boundary electrophoresis?
zone method for the purification of biomolecules
when was tools for DNA gel electrophoresis was developed?
1970
who developed the tools for DNA gel electrophoresis in 1970
(3 people)
Phil Sharp
Joe Sambook
Bill Sudgen
what did the inventors of the tool for DNA gel electrophoresis use?
agarose gel from seaweed
Why did Arne Tiselius develop electrophoresis?
to study serum proteins
what are the 3 parts of electrophoresis?
voltage
supporting medium
buffer system
this component of gel electrophoresis contains ions to conduct electricity
running buffer
type of electrophoresis in which supporting medium is a gel
gel electroforesis
the gel material acts as a __________ by retarding or completely obstructing the movement of macromolecules while allowing the smaller molecules to migrate freely
molecular sieve
T or F
gel electrophoresis is widely used to combine proteins and nucleic acids
F (used to separate)
what are the 2 set-ups of gel electrophoresis
vertical and horizontal gel electrophoresis
type of gel used for nucleic acids
agarose
type of gel used for smaller nucleic acid
polyacrylamide
gel structure held together by covalent cross-linking of acrylamide and N,N’-methylene bisacrylamide
polyacrylamide gel (PAG)
T or F
PAG can be used to separate DNA smaller than 1000 bp
F (less than 100 bp only)
component of discontinuous gel electrophoresis that contains a low percentage of acrylamide concentration and low pH
stocking gel
component of discontinuous gel electrophoresis that has a varying concentration of acrylamide and a higher pH
running / separating gel
T or F
in PAG electrophoresis DNA mobility observed in the gel (rate of migration) are dependent on the electrical field of strength used for the electrophoresis
F (it’s INDEPENDENT on the electrical field strength)
PAG electrophoresis depends on what?
3
size of analyte
pore size
concentration of acrylamide gel
T or F
concentration of gel is inversely proportional to the pore size
T
what should be the concentration and pore size needed for analysis of high molecular weight
low gel concentration, large pore
what should be the gel concentration and pore size for low molecular weight samples
high gel concentration, small pore
does PAG have high or low resolving power?
high
measures the ability of an electrophoretic system to separate DNA molecules of similar sizes’ effectiveness of separation
PAG
PAG or Agarose?
can accommodate larger quantities of DNA
PAG
T or F
Extremely turbid DNA may be recovered from PAG
F (pure)
T or F’
PAG are stable over the wide range of pH and temperature
T
T or F
PAG can withstand high voltage ingredients
T
PAG or Agarose
most effective in separating DNA fragments of varying sizes from 100 bp to 25 kb
Agarose (less than 100 bp lang for PAG)
Agarose gels are formed by suspending dry agarose in _______
aqueous buffer
at what temp is agarose heated?
100 degrees celsius
T or F
dissolved agarose gel produces a turbid mixture
F (clear, clearer than your future ganern)
At what temp does agarose solidify and form a gel
40-45 degrees celsius
T or F
linear carbohydrate polymer is composed of alternating units of agarobiose, consisting of repeating units of galactose and 3,6 anhydrogalactose
F (repeating agarobiose, alternating galactose and anhydrogalactose)
what is the charge of DNA and RNA molecules?
negative
Where does DNA pieces migrate to? anode or cathode?
anode (since their charge is negative, they’ll move to the positive pole)
A procedure that separates molecules based on their rate of movement through agarose gel under the influence of an electrical field
agarose gel elctrophoresis
familiarize the materials needed for agarose gel electrophoresis
electrophoresis chamber
agarose gel
gel casting tray
buffer
staining agent (dye)
comb
DNA ladder
sample
power supply
wire color that represents positive electrode (anode)
red
wire color that represents negative electrode (cathode)
black
material for agarose gel electrophoresis
available in variety of sizes and composed of UV transparent plastic (size depends on the samples used)
gel casting trays
material for agarose gel electrophoresis
placed in liquid agarose after it has been poured
comb
T or F
The comb can only be placed before pouring the liquid agarose
F (before or after)
what does the comb produce in the hardened agarose gel?
series of wells
what are the wells in the agarose for?
used to load / dispense the DNA
Material needed for agarose gel electrophoresis
solution that contains the right ions to conduct electricity
running buffer
What happens to DNA movement if water is used instead of running buffer
slow
Why is DNA movement slow if water is used instead of running buffer?
inefficient conduction of electricity because it has no ions
purpose of buffer
resist pH changes
most common buffer used in agarose gel electrophoresis
Tris buffer
this should be your 69th card if you’re a concrete sequential learner <33
wala lang skl
what does TAE stand for?
Tris acetate EDTA
what does TBE stand for?
Tris borate EDTA
what does EDTA inactivate?
DNA nucleases
what does DNA nucleases degrade?
nucleic acids
buffer mostly used for the analysis of larger samples (1500 bp and higher ; generates less current)
TAE
buffer mostly used for smaller nucleic acid fragments
TBE
in what for does buffers exist in?
neutral or cationic form
what is the pH of TAE
8
what is the pH of TBE?
8
what is the pH of Tris Phosphate EDTA buffer?
8
what is the pH of Tris-citrate (TCE) buffer
7
what is the other name for DNA ladder
Molecular weight marker
consists of known DNA sizes used to determine the size of an unknown DNA sample
DNA ladder
T or F
DNA ladder contains regularly spaced sized samples which when run on an agarose gel, it resembles a ladder
T
T or F
Each space on a DNA ladder represents a base pair
F (Each line)
T or F
the DNA ladder depends on the manufacturer
T
a set of standards used to identify the approximate size of molecule run on a gel during electrophoresis
DNA ladder
These are usually generated by restriction enzyme digestion of plasmid or bacteriophage DNA
DNA fragments
This runs alongside the DNA sample, usually on the first lane
DNA ladder
What should be added in the sample before being loaded on the gel?
loading dye
loading dye is composed of?
(2)
tracking dye and density agents
use this to familiarize density agents
glycerol
ficoll
sucrose
What is added to the sample to make it heavier in order for it to sink in the well
loading dye (because it contains a density agent)
This component of loading dye provides a visual gauge of the progress of electrophoresis
tracking dye
Does loading dye affect the separation of the sample?
no
what is the other term for loading dyes?
*blue text sa trans
Gel loading buffers
what are the two examples of tracking dye?
bromphenol blue
xylene cyanol
A compound that can adhere to DNA and gives off fluorescent light when excited by UV light
DNA stain
What is the standard concentration of DNA stain used in gels
0.5 - 1 ug / ml
This inserts itself between the base pairs in the double helix
DNA stain
most commonly used DNA stain
ethidium bromide
safer and non-mutagenic examples of DNA stain
GelRed
SYBR green
T or F
The color of the result is dependent on the type of sample, not the stain used
F (depends on the stain)
