Gel Electrophoresis Test

Explain how the analogy of the boy and mouse running through the forest can be used to explain how gel electrophoresis works. 

Dense forest - mouse will travel through faster since it is smaller and can fit between the trees

Similar to how gel electrophoresis works with the smaller particles moving faster than the bigger particles

What are the two bases upon which gel electrophoresis can separate out molecules?

Size: comparing it to the sizes of other relative particles

* smaller particles move more quickly through the gel than larger molecules

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Charge: negatively charged molecules move towards the positive pole of the unit

Why did we place the gel comb in the center of the gel during the molecular dye rainbow lab and on the end when working with DNA samples?

Teeth comb in the middle: (dye) unknown charge of the samples (can be attracted to the negative or positive electrode)

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Teeth comb on the end: (DNA) dna is negatively charged so it is attracted to the positive electrode. When the teeth comb are placed at the end, it can travel the farthest from the negative to the positive electrode.

What is the purpose of TAE in gel electrophoresis?

* provide ions that can carry electrical current across gel
* maintain relatively constant pH without damaging the DNA

What are the components of TAE buffer and what are they used for?

* Tris Base and Acetic Acid: maintains pH of 8-8.5 to protect DNA from hydrolysis (DNA breaks into fragments)
* EDTA: metal ion chelator to prevent metal ions from activating enzymes (DNase) from breaking down sample
* critical that buffer is used for both making the gel and running it

If I have a stock solution of 50X TAE, how much 50X TAE and distilled water would I need to make 750 mL of 1x TAE?

50\* *V = 1* \* 750

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15 mL of TAE

735 mL of distilled water

What is agarose? Where does it come from? Why do we use it to make our gels?

* polysaccharides purified from seaweed
* does not contain enzymes that could break down DNA
* neutral, effective method of separating nucleic acids

How does the percentage of a gel affect the size of DNA molecules it can separate?

Explain why this matters (you may use the analogy of the boy and the mouse again here)

* larger percentage: smaller molecules and DNA fragments
* smaller pore size/more trees → smaller the mouse can fit through
* smaller percentage: larger molecules and DNA fragments
* larger pore size/ fewer trees → bigger mice can travel through

Calculate the total grams of agarose you would need to prepare 4, 1.3% agarose gels

4 \*\* 11 ml \* 0.013 = 0.572 grams of agarose

How do you prepare an agarose gel?

1. Combine agarose and volume of TAE to flask (2x volume) and stir
2. Put in microwave and heat at 10 second intervals until bubbles
3. Let rest for 10 minutes or until the flask is cool to the touch
4. Add 1.0 uL of gelGreen for 10 mL of solution
5. Add to casting tray with correct comb and wait for 20 minutes to settle

What is the purpose of gel green?

Gel green is used to stain and visualize DNA samples in agarose gels during gel electrophoresis

What are the three purposes of loading dye?

1. provides a visible dye that helps you see what you are loading
2. dye moves through gel like small DNA molecules - can gauge how far the molecules have moved, prevent molecules from running off the gel
3. contains glycerol which is dense and will prevent DNA from diffusing out of the well and into the buffer