Gel Electrophoresis + PCR + Restriction Enzymes

Gel Electrophoresis

A lab procedure that is used to separate pieces of DNA by length.

What are the steps in gel electrophoresis?

Use micropipette to place DNA samples inside wells in gel square made of agarose. Place the gel in a liquid solution called buffer inside a gel electrophoresis chamber. Electric current is applied to the solution. DNA is negatively charged so it is attracted to the positive electrode and moves through the gel. After some time, the gel is removed from the box and the DNA fragments form bands on the gel that can be viewed by shining a UV light through the gel.

What is ethidium bromide and what does it do?

Sticks to the DNA and allows it to glow under UV light.

What does a buffer do?

Helps electricity flow through the gel.

What size DNA fragments move faster?

Small DNA fragments move faster through the thick matrix of the agarose gel.

Why is gel electrophoresis useful?

It can identify the size of an unknown DNA fragment.

What does the “ladder” or marker sample do?

Contains several known lengths of DNA.

What is the purpose of using a ladder in one lane on your gel?

You can estimate the unknown fragments that are in that lane.

Why do scientists need to use the PCR reaction?

Make millions of copies of the specific sequence they want to study.

What chemicals and reactants go into the tube before it goes into the PCR machine?

Small pieces of Rna called primers, water, nucleotides, the DNA strand, and an enzyme that copies DNA>

What do the RNA primers do in PCR?

Base-pair to the ends of the desired fragments and mark the specific segments that the scientists want to study and tell the reaction what DNA fragment to copy.

Why are the spare nucleotides needed in the reaction?

They are the building blocks that the DNA polymerase uses in order to create the PCR product.

How can scientists see what is in their reaction tube after the PCR reaction?

By using gel electrophoresis, the DNA fragments separate and it is easy to see them under UV light when using the addition of staining.

Restriction enzymes

Special enzymes harvested from unique bacteria that scientists use to cut DNA. They cut DNA but only at specific sequences.