Term
| Nucleic Acid-Specific Dyes |
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Definition
Used to visualize DNA products run via gel electrophoresis.
The two most common are Ethidium Bromide (an intercalating agent that stacks between N bases in dsDNA) and SyBr Green, which sits in the minor groove of DNA helices. |
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Term
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Definition
An intercalating agent when added to dsDNA. Will emit orange light when put under light of 300 nm when bound to DNA. Gels can be soaked in EtBr diluted in buffer or it can be added directly to the gel (although this may cause a bright band across the gel, obscuring results). A polaroid camera with appropriate filters can image the gel with fluorescent bands.
Carcinogenic. |
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Term
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Definition
SyBr Green I stains dsDNA, II stains ssDNA and ssRNA, and III stains RNA and DNA. Emits orange light when exposed to light. 25-100 times more sensitive than EtBr, and not carcinogenic. Is added to DNA samples, but this can lower sensitivity and slow sample migration down.
Not used widely because it needs new visualization filters. |
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Term
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Definition
| Used for protein visualization on gels. Gels are fixed with methanol and acetic acid before being impregnated with silver nitrate or ammoniacal silver in a weak acid. Under optimal pH, silver ions will interact with acidic or nucleophilic groups on the target and produce crystallization. The insoluble black silver salt precipitates when formaldehyde is added to the acid, or alkaline for silver nitrate. A biohazard and complicated because it must be watched and stopped at the right moment. Very sensitive. |
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Term
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Definition
| In gel electrophoresis the relationship between size and migration speed is simpler to resolve id DNA or RNA is single-stranded. Formamide prevents H-bonding and can be used to analyze single strands of nucleic acids in conjunction with urea or heat. |
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Term
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Definition
| Movement of charged electric particles via electric current. The ionized phosphates in DNA give it a negative charge, so it moves towards the positive anode. Because each nucleotide has 1 negative charge, there is a constant charge-to-mass ratio of molecules of different sizes. The migration speeds of DNA fragments are inversely proportional to their size. |
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Term
| Agarose Gel Concentrations |
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Definition
0.3% - 5000-60000 bp
0.6% - 1000-20000 bp
0.8% - 800-10000 bp
1.0% - 400-8000 bp
1.2% - 300-7000 bp
1.5% - 200-4000 bp
2.0% - 100-3000 bp |
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Term
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Definition
Gels provide resistance to molecule movement and prevent diffusion and convection currents. This assures that molecules form a band.
Gels must be unaffected by electrophoresis, cheap, and easy to prepare. |
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Term
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Definition
| Agarose is a linear polymer of agrobiose from seaweed. Can be bought ready made or as powder. Agarose concentration determines the gel pore sizes and what size of DNA fragments can be resolved. Higher concentrations are used for smaller fragments and vice versa. Concentrations over 5% impede migration and under 0.5% produce easily broken gels. |
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Term
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Definition
| A solvent flow towards the cathode in gel electrophoresis, resulting in slowed and distorted sample migration and smeared bands. |
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Term
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Definition
Alteration of agarose gel polymer length and helical parameters change its properties.
Low-melting point agarose can be used to run re-isolated resolved fragments from a gel.
Other type resolve larger or smaller fragments better. |
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Term
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Definition
| Agarose gel electrophoresis can't resolve DNA pieces over 50000 bp. Even at low agarose concentrations the pieces are impeded too much. Limiting mobility is reached when DNA molecules are so large that they can only move lengthwise through successive pores. |
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Term
| Pulsed Field Gel Electrophoresis |
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Definition
If pulses of current are applied to a gel in alternating dimensions, large DNA fragments can be resolved. Reorientation of the molecules helps them move. These methods take a long time because low voltage must be used and molecules take a long time to reorient themselves (usually 120 degrees). Larger fragments take longer switch intervals.
Used for bacterial typing for epidemiology. |
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Term
| Field Inversion Gel Electrophoresis |
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Definition
| Type of PFGE. The positive and negative electrodes are alternated during electrophoresis. DNA molecules reorient themselves 180 degrees, going periodically forwards and backwards. Can be performed in a normal gel matrix, but can only resolve pieces up to 2 megabases in length. |
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Term
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Definition
| Used to resolve full lengths of chromosome-sized DNA. In epidemiological bacterial typing, bacterial genomic DNA can be digested to yield a fragmented set unique to that species. Samples from different sources can be compared for similarities. |
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Term
| Polyacrylamide Gel Electrophoresis |
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Definition
Used to resolve small DNA fragments and ssDNA. Acrylamide polymerizes into a consistent matrix in conjunction with the cross-linker methylene bisacrylamide. Usually provided prepared since the powdered form is a neurotoxin.
Differences of 1 bp in a 1 kb molecule can be detected in a polyacrylamide gel, unlike agarose gels. |
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Term
| Polyacrylamide Gel Composition |
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Definition
Shown as total % concentration of the momomer (T) and the % of cross-linker (C). A 6% 19:1 gel has a T value of 6% and a C value of 1/20 (5%).
Pore size can be altered by changing C and T. Pore size decreases proportionally to increases in T. Minimum pore size is at C 5%. Normal C is 3.3% (29:1) for native and 5% (19:1) for DNA/RNA gels. |
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Term
| Capillary Electrophoresis |
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Definition
| A thin glass capillary of 300-100 cm in length with diameter of 25-100 um used to resolve molecules. The capillary is fused silica since this allows fluorescent light to shine through. A polyimide coating protects the silica except for a window. Hydroxyl ions from the silicone molecules dissociate and make a negative charge on the capillary walls, establishing an electro-osmotic flow when a current is applied. |
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Term
| Solution-Based Capillary Gel Electrophoresis |
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Definition
In the electro-osmotic flow generated in the capillary, small or negatively charged molecules migrate faster than large and positive ones. The best separation occurs when the proper buffer is used to ensure the proper charge on the analyte.
A buffer with high pH will completely ionize negative molecules. A low pH solution will completely protonate positive solutes. |
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Term
| Gel-Based Capillary Electrophoresis |
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Definition
| Nucleic acids don't separate in solution since their size and charge increase simultaneously. A polymer in the capillary will ensure that the nucleic acids migrate according to size, not charge. Secondary structure will affect speed, so the molecule must be single-stranded. Formamide is used to denature the nuclear acids in the buffer, and the capillary is held at a denaturing temperature (50-60 C) |
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Term
| Capillary Electrophoresis Sample Injection |
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Definition
| Samples enter the capillary via electrokinetic, hydrostatic, or pneumatic injection. Nucleic acids are generally taken up with electrokinetic injection, where an electrode close to the capillary end becomes briefly highly-positive to draw the sample up. The sample then migrates through the capillary, once current is established. |
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Term
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Definition
| Used to conduct the current and protect samples in gel electrophoresis. Buffers are made of a weak acid and its conjugate base in solution. Protons in the solution are taken up or released as solutes give off or absorb them, keeping the pH stable. |
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Term
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Definition
Equilibrium between an acid and base in buffer.
Ka = [H+][A-]/[HA]
pKa = -log Ka |
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Term
| Henderson-Hasselbach Equation |
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Definition
| Expresses the amount a pH will differ from the pKa of a buffer.
pH = pKa + log [basic form]\[acidic form]
If the two are in equal concentration, the pH=pKa. If not, and acidic form dominates, pH |
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Term
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Definition
| Buffers control the pH of a gel and protect samples from damage. Buffer ions carry the current, preventing severe pH fluctuations. The concentration must be high enough that both the acidic form and the basic form are present in such numbers to buffer the solution. High concentration also raises the gel system's conductivity and increases temperature, which must be compensated for with low voltage. |
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Term
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Definition
Tris Borate EDTA
-Greater buffering capacity
-Less exhaustible ions
-Slower DNA migration
-Not for postelectrophoretic isolation procedures
-Prone to precipitation
Tris Acetate EDTA
-Less buffering capacity
-Ion species more easily exhausted
-Faster DNA migration |
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Term
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Definition
| Nucleic acids migrate properly in a buffer that conducts electricity efficiently in relation to buffer capacity. Ions such as +2, -2, +3 with high charges move more quickly through a gel, increasing conductivity without increasing capacity. However, this results in too much current and quick buffer depletion. Tris base and borate remain partially uncharged at target pH, maintaining pH without high conductivity. |
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Term
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Definition
RNA is denatured with methylmuric hydroxide (MMH, toxic) and aldehydes to prevent base pairing.
Buffers used: 10 mM sodium triphosphate pH 7 and MOPS buffer.
Buffer needs to be re-circulated from anode to cathode to prevent pH drift. |
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Term
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Definition
Tracking dye and density agent (ficoll, glycerin, sucrose) are added to the sample before loading.
Tracking dyes monitor the progress of the run, migrating at specific speeds in certain gel concentrations. Do not affect sample separation,
Bromophenol blue and xylene cyanol green are often used. |
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