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32 Cards in this Set

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What is the definition of electrophoresis?

The separation of charged molecules by differences in their rate of migration in an electric field.

What is separation in electrophoresis based on?

Mobility of ionised compounds or particles dissolved in a conducting medium under the influence of an electric field

What is mobility?

Mobility, μ, is the average velocity with which ions movie in an applied electric field where the velocity is determined by two opposing forces

What are the four different components of an electrophoresis system?

1. Molecules to be separated


2. Support medium


3. Buffer system


4. DC power source

What are two common support media for proteins in electrophoresis?

1. Native gel (acrylamide or starch)


2. Denaturing (SDS) gel (acrylamide)

What are two common support media for nucleic acids in electrophoresis?

1. Agarose gel


2. Acrylamide gel

Name seven factors that influence mobility in electrophoresis

1. Molecular size (MW)


2. Molecular shape


3. Molecular charge




4. Electric field strength (V/cm)


5. Porosity of support medium (%S)


6. Conductivity of the buffer (k)


7. pH of the buffer

What is the significance of buffer pH in electrophoresis?

Affects charge of molecule

What is the effect on mobility as MW decreases?

Lower MW = higher mobility

What is the effect on mobility as field strength increases?

Increased field strength = higher mobility

Name two ways of increasing field strength

1. Increase voltage


2. Decrease length of gel

What is allowable field strength limited by?

Conductivity of the buffer. High conductivity = high current = heat.)

How does conductivity affect mobility?

High conductivity = higher mobility

What is the basis of separation of nucleic acids, and why?

Based on number of base pairs. Because Charge/BP is constant. Larger molecules move slower due to friction with gel.

What is the basis of separation of proteins, and why?

Charge varies as function of amino acid composition and buffer pH. Separation based on charge/MW. Exact combination of factors varies for each molecule.

Outline the conditions for the separation of DNA in agarose gels?

- 1.5x TAE Agarise gel


- TAE = 40mM Tris (pH buffer), 20mM acetic acid (pH regulator), 1mM EDTA (ion chelator)

How are nucleic acids stained?

- Ethidium bromide. DNA intercalator + UV fluorescence.

Why are denaturing gels used to separate proteins?

Too many separation parameters if using agarose gels.

Outline the process of separating proteins using denaturing gels

1. Proteins are heat denatured, this makes them all the same shape (linear). Disulphide bonds reduced using dithiothreitol or β-mercaptoethanol.


2. Proteins are coated with ionic detergent (SDS) which gives all molecules approximately the same overall negative charge.


3. Separation is therefore based on size (MW) alone.

What does SDS stand for?

Sodium dodecyl sulphate

Outline the conditions of protein separation

- Polyacrylamide gel, single percentage or variable gradient.


- Variable gradient allows for: separation of large molecules at top due to low percentage, and separation of small molecules at bottom due to high percentage)

What is capillary zone electrophoresis?

Electrophoresis carried out using narrow-bore capillaries (25-125μm internal diameter). It uses a very high electric field. The large SA:volume ratio allows for rapid dissipation of heat.

What are two advantages of CZE (capillary zone electrophoresis)?

1. Very short analysis times


2. High peak efficiency

Draw a basic schematic of CZE


What kind of compounds is CZE ideal for?

Compounds that present costly and time consuming challenges in chromatography. For example: proteins, peptides, nucleic acids, basic drugs, chiral compounds.

Do neutral compounds separate?

No

Do negatively-charged compounds travel towards the negative pole?

No, they travel towards positive pole.

How does electroosmotic flow arise?

Results from electrical double-layer that exists at the liquid-solid interface between capillary wall and bulk liquid.

How is electroosmotic flow different to pressure-driven liquid flow?

Plug-like, does not have parabolic flow where bulk liquid flows faster than that adjacent to capillary walls. This allows for high efficiency compound separation and high resolution of peaks.

How did Sanger sequencing help the human genome project?

It was the method used to produce the first human genome in 2001 (chromosome 22).

When was sanger sequencing most used?

1980s to the mid-2000s.

What was used to visualise the sequence in Sanger sequencing?

32P-ATP