Lab 11

Question 1

Gel Electrophoresis

Answer 1

Gel electrophoresis is how we can visualize DNA and determine the size of our DNA of interest.
- used to separate molecules such as DNA , RNA , and proteins based on their size and charge.

Question 2

DNA Gel Electrophoresis

Answer 2

the DNA is loaded into wells within a solid hydrated matrix. An electrical current is applied, and the negatively charged DNA molecules move through the gel towards the anode (+) end of the gel box.

DNA molecules migrate towards the positive electrode because of the negatively charged phosphates along the DNA backbone.

Larger molecules do not migrate as quickly as smaller molecules because of the greater frictional drag caused by the agarose matrix and because they must work their way through the pores in the matrix.

Question 3

Agarose gel

Answer 3

The gel used in DNA electrophoresis is composed of solubilized agarose. Extracted from seaweeds, agarose is a polysaccharide polymer.

When added to a liquid, heated and then cooled, the polysaccharide molecules form cross-links, creating a semi-solid matrix with pores.

Agarose gel electrophoresis separates DNA molecules based on size such that large molecules do not travel as far as small molecules. DNA

Question 4

Separation of Molecules

Answer 4

The gel acts like a sieve for DNA restricting movement of DNA through the gel.

Smaller molecules squeeze through the pores more easily so migrate faster through the gel compared to larger (longer) DNA molecules.

All DNA molecules of a given size travel at the same rate through the gel, forming a distinctive band of material.

Smaller DNA molecules (bands) are at the bottom of the gel, while larger molecules (bands) are near the top.

amount of time you run the gel also determines how well different bands are separated.
- careful not to run the gel for too long , other wise the smaller pieces of DNA will run off the end of the gel and into the buffer = lost forever

Circular molecules of DNA will migrate on a gel differently than linear molecules of the same size in base pairs.

Question 5

Undigested plasmid DNA from a mini-prep can exist in three different conformations

Answer 5

1. supercoiled circular plasmid DNA (wound up in a very tight ball) runs faster on a gel compared to a linear piece of DNA of the same size. Plasmid DNA carefully extracted should be observed primarily in this form.
2. nicked circular / relaxed circular plasmid in which one of the backbones of the DNA helix is broken thus preventing the formation of supercoiled DNA so the plasmid exists as a loose circle. Nicked circular plasmid DNA runs slower on a gel compared to a linear piece of DNA of the same size. Plasmid DNA that has been roughly extracted may run in this form.
3. linear plasmid DNA in which the plasmid has been linearized (both strands of the DNA helix have been cut) will run ‘true’ to the number of base pairs. Molecular weight markers are a series of known sizes of linear fragments that can be used to estimate the size of a cut fragment. Linear plasmids can result from a very rough extraction but also from digestion with a restriction enzyme.

Question 6

Loading Samples

Answer 6

loading dye is added to the DNA sample before it is loaded into the wells at the top of the gel.

• Loading dye contains bromophenol blue, an indicator dye, which gives the sample a visible blue color and allows you to track the progression of the dye through the gel. (Bromophenol blue will run as fast as a 500bp DNA fragment).
• dye also contains sucrose that increases the density of the DNA solution, allowing it to sink to the bottom of the wells and stay there during the loading procedure.

Question 7

Visualizing DNA in gels

Answer 7

A DNA dye is used to visualize DNA bands under UV light. Rather than adding it to the DNA samples, fluorescent dye is added to the gel before it is poured.

• most common dye used, called ethidium bromide, is a flat planar molecule that intercalates between the base pairs within the DNA molecule and fluoresces when exposed to ultraviolet (UV) light.
  –> (Ethidium bromide is a suspected carcinogen and must be handled with care. We will use an alternative dye, called Sybr-Safe, but will assume it is equally toxic.)

As the DNA runs through the gel, it is bound by the dye molecules. Once the gel has run long enough to allow for separation of DNA fragments, it is illuminated with a UV light source, and bands of DNA are highlighted by the fluorescing dye.