In its present form the DNA is crystalized and not able to travel through the agarose gel matrix. The DNA must be resuspended in solution before it can be run on a gel and analyzed by gel electrophoresis. You are going to pull the DNA out of the ethanol and transfer it back into an aqueous solution. The DNA will resuspend back into solution and no longer be visible.

Resuspending Your DNA

Each group should get the following materials from the front table:

• 2 small (1.5 or 2.0 mL) Eppendorf tubes
• 1 glass pipette hook
• 1 permanent marker
  
  

1. Label both the lid and the sides of a 2.0 mL Eppendorf tube with your initials, period, and "EtOH". Add 1 mL of 95% ethanol to the first tube. Leave the second tube empty for now.
   
   
2. Using a glass pipette hook, pull your DNA from your 15 mL tube and transfer it to the tube filled with 95% ethanol. Wiggle the DNA around in the ethanol for about 1 minute. (If the DNA falls off of the hook, close the cap and gently rock the tube back and forth.) The ethanol will cause some of the salts to precipitate away from the DNA. This will help to remove some of the contaminants.
   
   
3. Label both the lid and the sides of the second Eppendorf tube with your initials, period, and "DNA". Using the same glass hook, transfer your DNA from the tube filled with ethanol, to the clean, empty tube. Without touching the DNA, try to get the DNA to attach to side of the tube so you can remove the glass hook. If you can't get the DNA to disengage itself from the hook, it may be necessary to break the glass hook inside the tube. Ask your teacher for help.
   
   
4. Place your tube in a vacuum bell or under a bright light until there is no trace of ethanol in the tube. This will take at least 15-30 minutes.
   
   
5. Add 1 small drop (or 100 uL) of TE (or water) to the tube with your DNA. Gently flick the tube to mix. The TE buffer protects the DNA from enzymes which might break down the DNA. DNA can also be resuspended for short periods of time in water.
   
   
6. Leave the DNA sit in the tube for 3-4 days in order to completely resuspend into solution. The DNA needs to be suspended in solution in order to be used in other experiments including gel electrophoresis and PCR (polymerase chain reaction).
   
   
   

Science Education Connection Department of Biochemistry The University of Arizona Tuesday, January 14, 1997 warder@u.arizona.edu http://biology.arizona.edu/sciconn/lessons/vuturo/ All contents copyright © 1997. All rights reserved.