Electrophoresis

Question 1

What is electrophoresis used for

Answer 1

• techniques that seperates different molecules
• extensively used in the analysis of proteins and DNA

Question 2

How is electrophoresis done?

Answer 2

• technique involves placing a mxiture of molecules into wells cut into a gel and applying an electric field

Question 3

What doe sthe movement of charged molecules within the gel depend on?

Answer 3

• net charge
  
  • negatively charged molecules move toward the anode and postively charged molecules move towards the cathode(-) (cations move towards the cathode)
  • highly charged molc move faster than those with less overall charge
• size
  
  • smaller molecules move through the gel faster than larger molecules
• composition of the gel
  
  • common gels are polyacrylamide for proteins and agarose for DNA
  • the size of the ‘pores’ within the gel determines the speed with proteins and fragments of DNA move

Question 4

What are the difffernt positions on a micropipette?

Answer 4

• rest position
• first stopo
• second stop

Question 5

How is a micropipette filled

Answer 5

• fit the tip
  
  • press down and twist slightly to ensure an airtight seal
• hold the pipette in a vertical position
  
  • ppisj the plunger to the first stop
  • air equal to the volume of the setting is displaced
• immerse the tip into the sample
  
  • release the plunger back to the rest position
  • wait a second for the liquid top be sucked into the tip
  • volume of liquid in the tip will equal the volume of the setting of the micropipette
• *

Question 6

How is the sample added to the well?

Answer 6

• hold the micropipette using your other hand to support the hand holding the pipette
  
  • so its kept steady while guidng the tip into the well
• place tip at an angle against the well
• plush plunger the first stop, wait one second, then press plunger to second stop to expel all the liquid
  
  • if you press the plunger all the way, this will blow air into the well adn distrub the sample
• keep pressing the pipette plunger until the tip has been lifted out of the well
  
  • otherwise you might aspirate some of the sample from the well
• move the end of the tip away from the liquid
  
  • release the plunger to the rest position

Question 7

Why do you not insert the tip too deeply into the well?

Answer 7

• might puncture the well
• causing the smaple to leak underneath

Question 8

What is ethidium bromide used for > how is it used?

Answer 8

• stain DNA
• only visible with UV light
• also a mutagen
  
  • only used in advanced facilities