Gel Electrophoresis

Question 1

What is the technique of gel electrophoresis used for?

Answer 1

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

Question 2

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

Answer 2

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

Question 3

Describe the set-up of a gel electropheresis machine

Answer 3

• 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

Question 4

Describe the charge of DNA

Answer 4

The phosphate-backbone of DNA carries a negative charge

Question 5

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

Answer 5

• Will travel towards Positive electrode
• Opposite charges attract

Question 6

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

Agarose is made form seaweed derived material

Answer 6

The size of DNA molecules

Question 7

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

Answer 7

• 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)

Question 8

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

Answer 8

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

Question 9

What is the DNA Ladder

Can exist as RNA ladder as well

Answer 9

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

Question 10

Describe 1st step in a Gel Electrophoresis

Set-up

Answer 10

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

Question 11

Describe 2nd step in a Gel Electrophoresis

Solution added

Answer 11

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

Question 12

What is a buffer solution?

Answer 12

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

Question 13

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

Answer 13

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.

Question 14

Describe 2nd step in a Gel Electrophoresis

Movement of DNA particles

Answer 14

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

Question 15

Describe 3rd step in a Gel Electrophoresis

Stopping gel

Answer 15

• 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

Question 16

Describe 4th step in a Gel Electrophoresis

Comparing results

Answer 16

• 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.

Question 17

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?

Answer 17

• 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.

Question 18

List some purposed of Gel Electrophoresis

Answer 18

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