Agarose Gel Electrophoresis Flashcards
(36 cards)
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.
