MOL BIO LAB L8 (Semis- Electrophoresis) Flashcards by CHENNEDETTE RESYL MAGLANGIT

The movement of molecules by (DNA or RNA) and by (applying specific voltage) an electric current

Electrophoresis

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This can occur in air or solution or in a matrix to limit migration and contain the migrating material

Electrophoresis

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Each phosphate group on a nucleic acid polymer is ________ making the molecule negatively charged

ionized

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Under an electric current, DNA and RNA will migrate toward?

the positive pole (anode)

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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

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Provide resistance to the movement of molecules under the force of an electric current

Gel system

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Serve as a support medium for analysis of the separated components

Gel or Gel system

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Enumerate the Criteria for Best Matrix

Unaffected by electrophoresis
Simple to prepare
Amenable to modification

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Prevent diffusion and reduce convection currents so that the separated molecules form a defined group, or “band”.

Gel system

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Polymers that meet the criteria for best matrix

Agarose gel and Polyacrylamide

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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%

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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%.

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Reasons why gel strength of any concentration of agarose degrades.

Decrease over time
Exposure to chaotropic agents such as urea

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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

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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

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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)

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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)

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Works by alternating the positive and negative electrodes during electrophoresis

Field- inversion gel electrophoresis (FIGE)

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The simplest approach to this method (PFGE)

Field- inversion gel electrophoresis (FIGE)

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In this type of separation, the DNA goes periodically forward and backward

Field- inversion gel electrophoresis (FIGE)

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Used for applications that require the resolution of chromosome-sized fragments of DNA, such as in bacterial typing for epidemiological purposes.

Alternating-field electrophoresis

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This will yield a set of fragments that produce a band pattern specific to each type of organism

Enzymatic digestion of genomic DNA

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Requires a catalyst. Has higher resolution capacity for smaller fragments

Polyacrylamide Gels

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Acrylamide in combination with the cross-linker ____________, polymerizes into a matrix that has consistent resolution characteristics

methylene bisarcylamide

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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

1. High pH 2. 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

1. Tris-borate EDTA (TBE) 2. Tris-phosphate EDTA (TPE) 3. 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

1. Formamide 2. Urea 3. 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

1. Regular combs- horizontal gel 2. 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

1. Tracking Dye 2. Density agent

**Increases the density** of the sample as compared with the electrophoresis buffer

Density agent

Two Most Common Tracking Dye

1. Bromophenol blue 2. 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

1. Silver diamine 2. 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