PINES CITY COLLEGES

COLLEGE OF MEDICAL LABORATORY SCIENCE

2nd SEMESTER A.Y. 2022-2023
MOLECULAR BIOLOGY LAB

TITLE: ELECTROPHORESIS EQUIPMENTS

OBJECTIVES:

 Describe the general types of equipment used for electrophoresis and how samples are
introduced for electrophoretic separation

 Compare and contrast detection systems used in nucleic acid applications.

Electrophoresis Equipment
 Agarose gels are run horizontally, and polyacrylamide gels are run ________.
 Horizontal gels are run in acrylic containers called _________ or _____ that are divided into
two parts with a platform in the middle on which the gel rests.
 ___________ make up the electrodes in the gel compartments. The wires are connected to a
power source by __________ or connectors through the walls of the container.
 The gel in the box is submerged with electrophoresis _______ filling both compartments and
making a continuous system through which the current flows.
 The thickness of the gel and the volume of the buffer affect migration, so these parameters
should be kept constant for consistent results. As the gel is submerged through the loading
and electrophoresis process, horizontal gels are sometimes referred to as ____________.
 The power supply will deliver voltage, setting up a current that will run through the gel buffer
and the gel, carrying the charged sample through the matrix of the gel at a speed
corresponding to the charge/mass ratio of the sample molecules.
 Horizontal agarose gels are cast as square or rectangular slabs of varying size.
 The_________ of the gel solution will determine the ___________ of the gel.
 Agarose, supplied as a dry powder, is mixed at a certain percentage (w/v) with electrophoresis
buffer and heated on a heat block or by microwave to dissolve and melt the agarose.
 The molten agarose is cooled to 55 degree–65degree C, and a certain volume is poured into the
casting tray.
 A comb is then inserted into the top of the gel to create holes, or wells, in the gel into which
the sample will be loaded. The size of the teeth in the comb will determine the volume of
loaded sample and the number of teeth will determine the number of wells that are available in
the gel to receive samples. The gel is then allowed to cool, during which time it will solidify.
After the gel has polymerized, the comb is carefully removed and the gel is placed into the gel
box and submerged in electrophoresis buffer.

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  Vertical gel boxes have separate chambers that are connected by the gel itself.
 Electrodes are attached to the upper and lower buffer chambers to set up the current that
will run through the gel.
 The gel _____________ before filling the upper chamber with buffer.
 Some systems have a metal plate attached to the back of the gel to maintain constant
temperature across the gel.
 Maintaining constant temperature throughout the gel is more of a problem with __________
because the outer edges of the gel cool more than the center, slowing migration in the outer
lanes compared with lanes in the center of the gel. This is called “__________” because
similar-sized bands in the cooler outer lanes will migrate slower than comparable bands in the
inside lanes. Ensuring that there is no variation in temperature across the gel prevents gel
smiling from occurring.
 Vertical gel systems can range from large sequencing systems (35 cm x 26 cm) to mini-systems
(8 cm x10 cm).

Gel Loading
 Prior to loading the sample containing isolated nucleic acid onto the gel, __________ and a
__________ are added to the sample.
 The density agent __________ the density of the solution as compared with the
electrophoresis buffer.
Example: Ficoll, sucrose, or glycerol)
 When the sample solution is dispensed into the wells of the gel below the surface of the
buffer, it sinks into the well instead of floating away in the buffer.
 The tracking dyes are used to __________ the progress of the electrophoresis run.
 The dyes migrate at specific speeds in a given gel concentration and usually run ahead of the
smallest fragments of DNA.
 They are not associated with the sample DNA, and thus they do not affect the separation of
the sample DNA.
 The movement of the tracking dye is monitored, and when the tracking dye approaches the end
of the well electrophoresis is ________.
 _________________ is a tracking dye that is used for many applications.
 _________________ is another example of chromophores that are used as tracking dyes for
both agarose and polyacrylamide gels.
DETECTION SYSTEMS
Nucleic Acid–Specific Dyes
 Intercalating agents intercalate, or stack, between the nitrogen bases in double-stranded
nucleic acid.
Example: Ethidium bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (EtBr Under
excitation with ultraviolet light at 300 nm, EtBr in DNA emits visible light at 590 nm.
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  DNA separated in agarose or acrylamide and exposed to EtBR will emit ______ light when
illuminated by ultraviolet light at 300 nm.
 EtBr was the ___________ used dye in early DNA and RNA analyses. Care must be taken
in handling EtBr because it is ___________.
 After electrophoresis, the agarose or acrylamide gel can be soaked in a solution of 0.1–1–
mg/ml EtBr in running buffer.
 Alternatively, ___ can be added directly to the gel before polymerization or to the
running buffer. The latter two measures save time and allow visualization of the DNA
during the run. Dye added to the gel may form a bright front across the gel that could
mask informative bands. Dye added to the running buffer produces more consistent
_________.
 Some enclosed gel systems contain EtBr inside a plastic enclosed gel cassette, limiting
exposure and waste. After soaking or running in EtBr, the DNA illuminated with ultraviolet
light will appear as orange bands in the gel.
 ___________ is one of a set of stains introduced in 1995 as another type of nucleic acid–
specific dye system.
 SyBr green in association with DNA or RNA also emits light in the _______ range.
 SyBr green staining is 25–100 times _________ than EtBr.This is due, in part, to
background fluorescence from EtBr in agarose. A1x dilution of the manufacturer’s 10,000X
stock solution of SyBr green in TAE, TBE, or TE can be used in methods described for
EtBr.
 A 1/100 dilution of SyBr green can also be added directly to the DNA sample before
electrophoresis. DNA prestaining decreases the amount of dye required for DNA
visualization but ________ the sensitivity of detection and may, at higher DNA
concentrations, interfere with DNA migration through the gel.
 Because SyBr green is not an intercalating agent, it is not as _________.
 Scanning and photographic equipment optimized for EtBr would have to be modified for
optimal detection of the SyBr green stains.
 SyBr green is the preferred dye for _____________ methods.
Silver Stain
 After electrophoresis, the sample is fixed with _____________________.
 The gel is then impregnated with ______________________ solutions or _______________
in a weakly acid solution.
 Interaction of silver ions with acidic or nucleophilic groups on the target results in
___________ or deposition of metallic silver under optimal pH conditions.
 The insoluble ____________ salt precipitates upon introduction of ___________ in a weak
acid solution or alkaline solution for sliver nitrate.
 ______________ is best for _____ gels.
 Silver nitrate is considered to be more stable.

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  Silver staining avoids the hazards of the intercalators, but silver nitrate is itself also a
biohazard.
 Silver staining is more complicated than simple intercalation. Color development must be carefully
watched as the precipitate accumulates in order to stop the reaction once optimal signal is
reached.
 ________________ of the gel will result in high backgrounds and masking of results. The
increased sensitivity of this staining procedure makes up for its limitations.
 It is especially useful for _________ analysis and for detection of limiting amounts of product.

References:
 Molecular Cell Biology Ninth Editio ©2021 Harvey Lodish; Arnold Berk; Chris A. Kaiser;
Monty Krieger; Anthony Bretscher; Hidde Ploegh; Kelsey C. Martin; Michael Yaffe;
Angelika Amon
 Molecular Biology Made Simple and Fun, Third Edition 3rd Edition by David P. Clark

Education is the passport to the future, for tomorrow belongs to those who prepare for it
today.
-Malcolm X-

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