Agarose Gel Electrophoresis Flashcards
On what basis does electrophoresis separate molecules?
Charge, size, and shape.
Electrophoresis is useful in separating charged biomolecules like…
DNA, RNA, and proteins. (Also dye mixtures.)
The gel is made of…powder from…
Agarose (powder) from seaweed.
What do you put the gel in?
A buffer-filled chamber containing electrodes.
What do you mix with samples to make them more dense?
A dense solution such as glycerol or sucrose.
Why do the samples need to be dense?
They must be denser than the buffer solution so that they can sink into the wells.
What is the purpose of the buffer in the gel chamber?
It completes an electric circuit between electrodes.
Negatively charged molecules (anions) will migrate to…
The positive electrode (anode, red).
Positively charged molecules (cations) will migrate to…
The negative electrode (cathode, black).
What is the purpose of the buffer salts?
They make the water a better conductor of electricity and control the pH.
What characteristic of agarose makes it useful for electrophoresis?
It contains microscopic pores that act as molecular sieves.
If two molecules have the same charge and weight, which one will be faster?
The one with a more compact shape. (Smaller molecules will move faster and further.)
What variables influence the way the charge, size, and shape of molecules interact with each other during electrophoresis?
Buffer conditions, structure and composition, gel concentrations, voltage.
One microliter=? milliliters
0.001 mL
1 mL=? microliters
1,000 microliters
How should you load the microcentrifuge?
With the rotor balanced (for example, you cannot spin just one tube) to prevent damage to the motor.
A microliter is a…of a liter
Millionth.
P-10 range and dial at 1.2 microliters (cap=white)
The range is 0.5-10 microliters. The dial set at 1.2 looks like this:
0
1
2
P-200 range and dial at 35 microliters (cap=gold)
20-200 microliters. Dial at 35:
0
3
5
P-20 range and dial at 15 microliters (cap=lemon yellow)
2-20 microliters. Dial at 15:
1
5
0
P-1000 range and dial at 550 microliters (cap= blue)
200-1000 microliters. Dial at 550:
0
5
5
Directions for the correct use of the micropipets
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.
What is the purpose of the buffer liquid?
It controls pH, stabilizes the process, and lets electricity move through.
Before dispensing liquid, what should you do with the box?
Move it close to the power supply.
Instructions after arranging the tubes in the microcentrifuge
Close the lid; let it run for 3 seconds. Open only after stopped.
What is the charge of DNA? Having a charge allows DNA to work with…
Negative, electrophoresis.
Hydrogen bonds are one of the (stronger/weaker) types of bonds.
Weaker.
How many chromosomes do humans have?
46 total; 23 from the sperm and 23 from the egg.
What are chromosomes?
Molecules of DNA.
What can you use to cut DNA in specific places?
Restriction enzymes.
What end do you start the DNA at during electrophoresis? Why?
The negative end (cathode, black), because DNA is negatively charged and will move across to the positive side.
What is the purpose of the stain?
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.
Why do you add dye before and after to the DNA?
So that the DNA is visible.
What can you do with DNA fingerprinting/profiling?
You can investigate whose DNA it is by matching it.
What is the most common DNA test?
DNA paternity testing.
Why is it important for the buffer to maintain pH?
Because pH is important for the charge and stability of many molecules and so you don’t damage the substance.
