Week 9: Gel Electrophoresis

What is the procedure for conducting gel electrophoresis?

1. Prepare agarose solution, let gel set. 2. Add loading dye to PCR samples. 3. Set up electrophoresis chamber and fill with buffer. 4. Load PCR samples into the wells. 5. Add DNA ladder to last well. 6. Close chamber and turn on. 7. When DNA has migrated at least 6 cm you may read it.

What kind of gel was used in this lab?

Agarose

What is the function of agarose?

Agarose serves as a gel that DNA fragments must move through. The agarose allows the DNA fragments to be separated based on size.

What does each band represent in gel electrophoresis compared to the DNA ladder?

All of the DNA fragments of identical legnth

What is the purpose of the buffer in gel electrophoresis?

Allows the agarose solution to carry a charge

What is the positive pole called in gel electrophoresis?

Anode

Why did the transformed bacteria only fluoresce in the presence of arabinose, even though the results of your PCR and gel electrophoresis indicate the GFP gene was present in all transformed colonies?

Arabinose is the activator for GFP, even if the GFP gene is present but arabinose is not then the GFP cannot fluoresce.

Why can smaller fragments of DNA move farther than larger fragments in the agarose gel?

Because the small fragments can make their way through the pores easier than the large ones.

Why should you omit the 23 kb band when constructing your best fit line in this run?

Because without it you get a line of best fit that is more accurate.

What is the negative pole called in gel electrophoresis?

Cathode

What does the buffer do in this lab?

Chelates Mg²⁺ to protect DNA from enzymatic degradation by DNAse.

What are base pairs?

Corresponding nucleotides. AT, CG

In the electrical field, what direction (+ or -) does DNA migrate? Why?

DNA is negatively charged because of the phosphate backbone so it migrates toward the positive pole.

What is on the x axis in the graph?

Distance from wells that DNA fragment traveled.

The movement of suspended particles through a medium

Electrophoresis

What is on the y axis in the graph in the gel electrophoresis experiment?

Fragment length (base pairs)

Definition: somewhat dense, jelly-like substance

Gel

Why is loading dye added to the agarose solution?

It allows you to visually see movement of DNA

What is the function of the buffer?

It supplies ions. Distilled water has no ions to carry charge, Acetate can carry the charge. This helps Mg ions migrate through the agarose.

Does the dye actually bind to the DNA?

No, it just migrates slightly in front of it.

Do smaller fragments move farther through the agarose gel or larger fragments?

Smaller fragments migrate farthest

The dye we used in the gel electrophoresis lab is called what?

Sybr safe

What is the function of sybr safe?

Sybr safe allows you to see how far DNA fragments have migrated toward the positive pole

PCR products from which of the plates tested positive for the presence of GFP protein?

The 2nd and 3rd wells.

What would happen if you removed the Mg?

The DNA activity ceases because the DNA would be broken down into small pieces.

What would have happened in the electrophoresis if the electrodes had been reversed?

The DNA won't move because the DNA has a negative charge and is attracted to positive charge. If you reverse them and already have the negative charge next to the positive charge, the DNA has reached a place where it is satisfied.

What is the function of UV light?

The UV light allows you to see how far the DNA fragments have travelled.

DNA isolated and amplified from bacteria on LB/amp plates that DID NOT glow tested positive for the presence of the pGLO gene. How can you explain that the gene was present, however no GFP was being produced?

The plate tested positive for pGLO gene but didn't show it because arabinose wasn't present and arabinose allows for the gene to be expressed

What would have happened in the electrophoresis if the gel chamber had been filled with water instead of gel buffer?

There are no ions in water, therefore ability of molecules to migrate though gel is lacking. If you add buffer it has ions in it and the electricity can migrate through the buffer, taking the DNA fragments with it.

What buffer was used in this lab?

Tris-EDTA buffer

Gel used in electrophoresis

agarose