Agarose Gel Electrophoresis

Question 1

On what basis does electrophoresis separate molecules?

Answer 1

Charge, size, and shape.

Question 2

Electrophoresis is useful in separating charged biomolecules like…

Answer 2

DNA, RNA, and proteins. (Also dye mixtures.)

Question 3

The gel is made of…powder from…

Answer 3

Agarose (powder) from seaweed.

Question 4

What do you put the gel in?

Answer 4

A buffer-filled chamber containing electrodes.

Question 5

What do you mix with samples to make them more dense?

Answer 5

A dense solution such as glycerol or sucrose.

Question 6

Why do the samples need to be dense?

Answer 6

They must be denser than the buffer solution so that they can sink into the wells.

Question 7

What is the purpose of the buffer in the gel chamber?

Answer 7

It completes an electric circuit between electrodes.

Question 8

Negatively charged molecules (anions) will migrate to…

Answer 8

The positive electrode (anode, red).

Question 9

Positively charged molecules (cations) will migrate to…

Answer 9

The negative electrode (cathode, black).

Question 10

What is the purpose of the buffer salts?

Answer 10

They make the water a better conductor of electricity and control the pH.

Question 11

What characteristic of agarose makes it useful for electrophoresis?

Answer 11

It contains microscopic pores that act as molecular sieves.

Question 12

If two molecules have the same charge and weight, which one will be faster?

Answer 12

The one with a more compact shape. (Smaller molecules will move faster and further.)

Question 13

What variables influence the way the charge, size, and shape of molecules interact with each other during electrophoresis?

Answer 13

Buffer conditions, structure and composition, gel concentrations, voltage.

Question 14

One microliter=? milliliters

Answer 14

0.001 mL

Question 15

1 mL=? microliters

Answer 15

1,000 microliters

Question 16

How should you load the microcentrifuge?

Answer 16

With the rotor balanced (for example, you cannot spin just one tube) to prevent damage to the motor.

Question 17

A microliter is a…of a liter

Answer 17

Millionth.

Question 18

P-10 range and dial at 1.2 microliters (cap=white)

Answer 18

The range is 0.5-10 microliters. The dial set at 1.2 looks like this:
0
1
2

Question 19

P-200 range and dial at 35 microliters (cap=gold)

Answer 19

20-200 microliters. Dial at 35:
0
3
5

Question 20

P-20 range and dial at 15 microliters (cap=lemon yellow)

Answer 20

2-20 microliters. Dial at 15:
1
5
0

Question 21

P-1000 range and dial at 550 microliters (cap= blue)

Answer 21

200-1000 microliters. Dial at 550:
0
5
5

Question 22

Directions for the correct use of the micropipets

Answer 22

Only set volumes within the range so you don’t break it or get inaccurate measurements, always use a disposable tip to prevent cross-contamination, keep the pipet in a vertical position when it has liquid to prevent liquid from leaking back into the piston, and use your thumb to control the rate at which the plunger rises because letting it snap back can damage piston and dispense inaccurate volumes.

Question 23

What is the purpose of the buffer liquid?

Answer 23

It controls pH, stabilizes the process, and lets electricity move through.

Question 24

Before dispensing liquid, what should you do with the box?

Answer 24

Move it close to the power supply.

Question 25

Instructions after arranging the tubes in the microcentrifuge

Answer 25

Close the lid; let it run for 3 seconds. Open only after stopped.

Question 26

What is the charge of DNA? Having a charge allows DNA to work with…

Answer 26

Negative, electrophoresis.

Question 27

Hydrogen bonds are one of the (stronger/weaker) types of bonds.

Answer 27

Weaker.

Question 28

How many chromosomes do humans have?

Answer 28

46 total; 23 from the sperm and 23 from the egg.

Question 29

What are chromosomes?

Answer 29

Molecules of DNA.

Question 30

What can you use to cut DNA in specific places?

Answer 30

Restriction enzymes.

Question 31

What end do you start the DNA at during electrophoresis? Why?

Answer 31

The negative end (cathode, black), because DNA is negatively charged and will move across to the positive side.

Question 32

What is the purpose of the stain?

Answer 32

You put it in before the DNA so you can track the progress of the DNA. If the dye is near the end of the box, the DNA likely is, too.

Question 33

Why do you add dye before and after to the DNA?

Answer 33

So that the DNA is visible.

Question 34

What can you do with DNA fingerprinting/profiling?

Answer 34

You can investigate whose DNA it is by matching it.

Question 35

What is the most common DNA test?

Answer 35

DNA paternity testing.

Question 36

Why is it important for the buffer to maintain pH?

Answer 36

Because pH is important for the charge and stability of many molecules and so you don’t damage the substance.