Gel Electrophoresis Flashcards

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1
Q

What is the technique of gel electrophoresis used for?

A

Separate DNA strands (sometimes proteins) according to their size, charge and shape

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2
Q

What process is usually carried out before gel electrophoresis and why?

A
  • PCR (Polymerase Chain reaction)
  • To create enough DNA to be tested in gel electrophoresis
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3
Q

Describe the set-up of a gel electropheresis machine

A
  • Connected to a power supply
  • Has a negative electrode (negatively charged end) and a positive electrode (positively charged end)
  • Has wells near negative electrode
  • Gel contains agarose
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4
Q

Describe the charge of DNA

A

The phosphate-backbone of DNA carries a negative charge

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5
Q

Which direction will DNA travel in a gel electrophoresis and why?

A
  • Will travel towards Positive electrode
  • Opposite charges attract
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6
Q

What determines how far DNA strands travel through the agarose gel?

Agarose is made form seaweed derived material

A

The size of DNA molecules

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7
Q

How does the agarose in the gel separate the DNA strands by size?

A
  • Agarose creates a porous gel
  • Shorter DNA fragements travel fastest (can go through holes/pores quicker)
  • Larger/longer fragmnets move slowest (hard to fit through pores quickly)
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8
Q

What two factors of a Gel elcterophoresis set-up can be changed to affect speed and separation of DNA strands?

A
  • Voltage/power used - creates stronger charge in electrodes
  • Viscosity/Concentration of agarose - Creates gel more or less porous
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9
Q

What is the DNA Ladder

Can exist as RNA ladder as well

A
  • A mixture of DNA segments with varying lengths.
  • All lengths of these varying fragments are known.
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10
Q

Describe 1st step in a Gel Electrophoresis

Set-up

A
  • Set-up of electrophoresis chamberis finished and power turned on
  • Using micropipette DNA is loaded into wells
  • Well 1 contains DNA Ladder
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11
Q

Describe 2nd step in a Gel Electrophoresis

Solution added

A
  • A buffer solution is added.
  • A stain/dye is added to chamber to stain/colour DNA fragments.
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12
Q

What is a buffer solution?

A

Solution containing ions. Allows the electric charge to run through electrophoresis chamber.

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13
Q

What are the 3 ways to stain/colour/label DNA in an Electrophoresis chamber. Give breif detail.

A
  1. Methylene blue - visible as blue bands. Added after gel is complete.
  2. Fluorescent stains - Seen under UV light, illuminates DNA as pink colour. Can be added before or after gel has been run
  3. Labelled probes - Visible particles that bind to specifc DNA fragments. Probes are manufactured to be specific to certain DNA segments.
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14
Q

Describe 2nd step in a Gel Electrophoresis

Movement of DNA particles

A
  • DNA begins travelling to positive electrode.
  • Smaller particles travel quicker as they can get through porous gel quicker.
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15
Q

Describe 3rd step in a Gel Electrophoresis

Stopping gel

A
  • Power in gel is stopped after some time (about 20 minutes).
  • Each well’s lane now has parallel bands of DNA fragments that have travelled a certain distance
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16
Q

Describe 4th step in a Gel Electrophoresis

Comparing results

A
  • Bands of unknown DNA fragments in the lanes are compared to bands with known length in DNA ladder.
  • Length of DNA segments is then estimated.
17
Q

If a batch of DNA fragments with varying length are out through a gel electrophoresis chamber, but all fragment lengths were relatively short, what would you do to gel and why?

A
  • Increase percentage of agarose in gel
  • Creates more porous gel with smaller pores.
  • Thus, separates smaller fragments more effectively.

Opposite for batch of large DNA fragments.

18
Q

List some purposed of Gel Electrophoresis

A
  • Forensic Investigation
  • DNA profiling
  • Examining disease
  • Paternity disputes