MOL BIO LAB L8 (Semis- Electrophoresis) Flashcards
The movement of molecules by (DNA or RNA) and by (applying specific voltage) an electric current
Electrophoresis
This can occur in air or solution or in a matrix to limit migration and contain the migrating material
Electrophoresis
Each phosphate group on a nucleic acid polymer is ________ making the molecule negatively charged
ionized
Under an electric current, DNA and RNA will migrate toward?
the positive pole (anode)
In a matrix of these gels, migration under the pull of the current is impeded, depending on the size of the molecules and the spaces in the gel matrix
Agarose and Polyacrylamide
Provide resistance to the movement of molecules under the force of an electric current
Gel system
Serve as a support medium for analysis of the separated components
Gel or Gel system
Enumerate the Criteria for Best Matrix
- Unaffected by electrophoresis
- Simple to prepare
- Amenable to modification
Prevent diffusion and reduce convection currents so that the separated molecules form a defined group, or “band”.
Gel system
Polymers that meet the criteria for best matrix
Agarose gel and Polyacrylamide
Q1: Small pieces of DNA (________ [bp]) are resolved on more concentrated agarose gels
Q2: How many percent of agarose gel is applied for small pieces of DNA?
Q1: more; 50 to 500 base pairs
Q2: 2% to 3%
Q1: Larger fragments of DNA (____ to _____) are best resolved
in _____ agarose concentrations.
Q2: How many percent of agarose gel is applied for larger fragments of DNA?
Q1: 2,000 to 50,000; lower
Q2: 0.5% to 1%.
Reasons why gel strength of any concentration of agarose degrades.
- Decrease over time
- Exposure to chaotropic agents such as urea
Agarose concentrations above 5% and below 0.5% are not practical. Because high concentration agarose will __________, whereas very low concentrations produce a _________ that is easily broken
impede migration; weak gel
Q1: Used for very large pieces of DNA and bacterial typing for epidemiological purposes
Q2: How many base pairs?
Q1: Pulse- Field Gel Electrophoresis
Q2: 50,000 to 250,000 base pairs
Enumerate all the examples of pulse-field gel configurations
- Field- inversion gel electrophoresis (FIGE)
- Transverse alternating-field electrophoresis (TAFE)
- Rotating gel electrophoresis (RGE)
- Contour- clamped homogeneous electric field (CHEF)
For these very large DNA molecules, pulses of current applied to the gel in alternating dimensions enhance migration. This process is called?
Pulsed-field Gel Electrophoresis (PFGE)
Works by alternating the positive and negative electrodes during electrophoresis
Field- inversion gel electrophoresis (FIGE)
The simplest approach to this method (PFGE)
Field- inversion gel electrophoresis (FIGE)
In this type of separation, the DNA goes periodically forward and backward
Field- inversion gel electrophoresis (FIGE)
Used for applications that require the resolution of chromosome-sized fragments of DNA, such as in bacterial typing for epidemiological purposes.
Alternating-field electrophoresis
This will yield a set of fragments that produce a band pattern specific to each type of organism
Enzymatic digestion of genomic DNA
Requires a catalyst. Has higher resolution capacity for smaller fragments
Polyacrylamide Gels
Acrylamide in combination with the cross-linker ____________, polymerizes into a matrix that has consistent resolution characteristics
methylene bisarcylamide
Natural polymer from living organisms
Agarose
Why does the Polyacrylamide gel have precise control of the polymer properties and higher resolution than can be achieved with agarose?
Because it is a synthetic material
Polymerizes upon cooling
Agarose gel
Catalysts of polyacrylamide gel can be?
Nucleating agents
The two catalysts of Polyacrylamide gel
- Ammonium persulfate (APS)
- N, N, N’, N’ - tetramethylethylenediamine (TEMED) or light activation
Produces free oxygen radicals in the presence of TEMED to drive the polymerization mechanism
Ammonium persulfate (APS)
If light activation is used, free radicals are generated by a photochemical process using?
Riboflavin plus TEMED
In light activation, this inhibits the polymerization process
Excess oxygen
In light activation, this is done before the addition of the nucleating agents.
De-aeration or removal of air
The main advantage of polyacrylamide over
agarose
higher resolution capability of polyacrylamide for small fragments.
With single-base resolution, polyacrylamide gels are used for:
- nucleic acid sequencing
- mutation analyses
- nuclease protection assays
- other applications requiring high resolution of nucleic acids
Separates particles by size and charge
Capillary electrophoresis
In Capillary Electrophoresis, if the size is small, it migrates ______, whereas if it is large, it migrates _____.
faster; slower
In Capillary Electrophoresis, if it is negatively charge, it migrates _____, whereas if it is positively charged, it migrates _____.
fast; slow
__________ charged molecules are completely ionized at high pH whereas
___________ charged solutes are completely protonated in low-pH buffers
negatively; positively
To simplify:
Small and negatively charge particles- fast migration
Large and positively charge particles- slow migration
Give the pH: Low or High
1. Negatively charged
2. Positively charged
- High pH
- Low pH
Identify:
- Size and charge (charge/mass ratio)
- Increased sensitivity and immediate detection
Capillary Electrophoresis
These do not separate well in solution.
Nucleic acids (DNA & RNA)
Introducing a _____ inside the capillary establishes resolution by impeding nucleic acid migration according to size more than charge
polymer
In capillary electrophoresis, It is important that the nucleic acid be _________ so that it will be separated according to its size because the secondary structure will affect the migration speed
completely denatured (single stranded)
Advantages of Capillary system
increased sensitivity
and immediate detection
Carry the current and protect the samples during electrophoresis
Buffer system
Buffer most commonly used for DNA
Tris-Buffer
Types of Tris-Buffers
- Tris-borate EDTA (TBE)
- Tris-phosphate EDTA (TPE)
- Tris-acetate EDTA (TAE)
Has a greater buffering capacity than TAE.
Tris-borate EDTA (TBE)
Ion species are more easily exhausted in this Tri-buffer during extended or high-voltage electrophoresis
Tris- acetate EDTA (TAE)
DNA will migrate twice as fast in ____ than in _____ in a constant current
TAE; TBE
Modify sample molecules in ways that affect their migration
Buffer Additives
Examples of Buffer Additives
- Formamide
- Urea
- Detergents
Break hydrogen bonds between complementary strands or within the small strand of DNA or RNA
Denaturing agents (Formamide & Urea)
These gel systems maintain this conformation such that intrachain hybridization (folding) of the nucleic acid molecules does not affect migration speeds, and separation occurs strictly according to the size or length of
the molecule.
Urea and heat in the gel systems
Hinder complementary
sequences from reannealing
Formamide and heat
Reacts with amino groups on the RNA to prevent base pairing between complementary nucleotides and with aldehyde
Denaturing Agents (RNA)
methylmercuric hydroxide (MMH)
These are run in acrylic gel boxes or baths that are divided into two parts, with a platform
Horizontal gels
The thickness of the gel and volume of the buffer affect the _____, therefore the migration of the sample.
Current
This fills both compartments and makes a continuous system through which the current flows, this is also where the gel is submerged
Electrophoresis buffer
These parameters are kept constant for consistent results
thickness of the gel and volume of the buffer
Horizontal gels are sometimes referred to as ?
submarine gels
Volume of the gel solution will determine the ________ of the gel
thickness
Agarose is mixed at a certain percentage (w/v) with electrophoresis buffer and heated on a ______ or by _______to dissolve and melt the agarose
heat block; microwave
Molten agarose is cooled to between?
55C and 65C
Inserted into the top of the gel to create holes, or wells, in the gel into which the sample will be loaded
Comb
Statement 1: The size of the teeth in the comb will determine the _______ of the well for the sample,
Stament 2: The number of teeth in the comb will determine the __________ that are available in the gel to receive the samples
Statement 1: capacity
Statement 2: number of wells
2 Types of Combs
- Regular combs- horizontal gel
- Sharkstooth combs - vertical gel
These are cast between glass plates that are separated by spacers.
Vertical gels
The spacers determine the thickness of the gel, ranging from ____ to ____
0.05 to 4 mm
The bottom of the vertical gel is secured by ____ or by a _____ in specially designed gel casting trays.
tape; gasket
After the addition of catalyst and nucleating agents, the liquid acrylamide is poured or forced between the glass plates with a?
pipet or syringe
This is then placed on the top of the gel
comb
For light-activated polymerization, the gel between the glass plates is exposed to a?
light source
During light-activated polymerization, it is important to prevent air from getting into the gel or beneath the comb, why?
Bubbles will form discontinuities in the gel and oxygen will inhibit the polymerization of the acrylamide
The comb is of a thickness equal to that of the _____ so that the gel will be the same thickness throughout.
spacers
____________ is usually polyacrylamide while ___________ could either be agarose or polyacrylamide gels
Vertical gel; horizontal gel
Used to monitor the progress of the electrophoresis run
Tracking dye
The dyes migrate at specific speeds in a given gel concentration and usually run ahead of the smallest fragments of DNA
Tracking dye
Examples of density agents
ficoll, sucrose or glycerol
Gel loading is composed of
- Tracking Dye
- Density agent
Increases the density of the sample as compared with the electrophoresis buffer
Density agent
Two Most Common Tracking Dye
- Bromophenol blue
- Xylene cyanol green
A tracking dye that is used for many applications
Bromophenol blue
Another of the chromophores used as tracking dye for both agarose and polyacrylamide gels
Xylene cyanol green
Electrophoresis is terminated when?
When tracking dye approaches the end of the gel or the desired distance
Agents used most frequently for visualization of bands after electrophoresis
- Fluorescent dyes
- Silver stain
Both are associated with nucleic acid
Most widely used dye in early DNA and RNA analyses
Ethidium Bromide
EtBr is excited by UV light at ______ and in DNA emits visible light at ______
300 nm; 590 nm
DNA separated in agarose or acrylamide and exposed to EtBr will emit ____ light when illuminated at _____
orange; 300 nm
After electrophoresis, the agarose or acrylamide gel is soaked in a solution of _________ in running buffer (TAE, TBE, or TPE) or in TE.
0.1- to 1 mg/mL EtBr
Q1: Used for dsDNA
Q2: Used for ssDNA, RNA
Q1: SYBR Green I
Q2: SYBR Green II
This differs from EtBr in that it does not intercalate between bases; it sits in the minor groove of the double helix
SYBR Green I
SYBR green in association with DNA or RNA also emits light in the _____ range at ______nm.
orange; 522 nm
In agarose gel electrophoresis, SYBR staining is _______ times more sensitive than EtBr
25 to 100 times
This can also be added directly to the DNA sample before electrophoresis
SYBR green
This decreases the amount of dye required for DNA visualization but lowers the sensitivity of detection and may, at higher DNA concentrations, interfere with DNA migration through the gel
DNA prestaining (SYBR green)
Good for real time PCR
SYBR Green
Because ______ is not an intercalating agent, it is not as mutagenic and is therefore safer to use.
SYBR green
In SYBR Gold, fluorescence increase more than _____ - fold upon binding to double or single-stranded DNA or to RNA
1000- fold
Types of silver stains
- Silver diamine
- Silver nitrate
Like SYBR green, SYBR gold is excited by UV light at ______ wavelength
300 nm
A sensitive staining system originally developed for protein visualization
Silver Stain
When using silver stain, after electrophoresis, the sample is fixed with?
Methanol and acetic acid
When using silver stain, after the sample has been fixed, the gel is then impregnated with _______ in a weakly acid solution
ammoniacal silver (silver diamine) solutions or silver nitrate
Interaction of silver ions with acidic or nucleophilic groups on the target results in ____________, under optimal pH conditions
crystallization or deposition of metallic silver
This precipitates upon introduction of formaldehyde in a weak acid solution, or alkaline solution for silver nitrate
Insoluble black silver salt
Best used for thick gels
Silver diamine
Considered to be more stable (A silver stain)
Silver nitrate
These are especially useful for protein analysis and for detection of limiting amounts of product/ amplicons
Silver stains (silver diamine & silver nitrate)
SYBR Gold emits light at?
537 nm
