Download DNA extraction protocol

DNA Extraction Lab
Purpose: To provide you with the opportunity to isolate and observe DNA. You will gain
an understanding of how a fractionation procedure is carried out, the roles of each
substance involved and how easy it is to isolate DNA.
Background: The process of isolating DNA from a cell is the first step for many
laboratory procedures in biotechnology. One must be able to separate the DNA from the
unwanted substances of the cell gently enough so that the DNA is not broken up or
shredded.
A solution is made of check cells treated with salt, buffer and detergent (SDS). The salt
shields the negative phosphate end of DNA which allow these ends to come closer so
they can precipitate out of a cold alcohol solution. The detergent causes the cell
membrane to breakdown by emulsifying the lipids and proteins of the cell and disrupting
the polar interactions that hold the cell membrane together. The detergent then forms
complexes with these lipids and proteins, causing them to precipitate out of solution. The
buffering agent maintains the pH of the solution so that the DNA stays stable.
Collectively, the salt solution and detergent are referred to as the lysing or
homogenization buffer.
Protease, an enzyme that digests proteins, is added to remove proteins bound to the DNA
and to destroy cellular enzymes that would digest the DNA.
Materials and Equipment
3ml water (in 15ml tube)
protease solution
lysis buffer
ice cold ethanol
3 disposable pipets
Procedure
1. Obtain 3ml of water and label with your initials.
2. Gently chew the insides of your checks of 30 seconds. DO NOT draw blood!!
3. Take the water from the 15ml tube into your mouth. And swish the water around
vigorously for 30 seconds.
4. Expel the liquid back into the 15ml tube (there are also some small cups available to
help facilitate the transfer.
5. Add 2ml of lysis buffer to your tube.
6. Place the cap on the tube and gently invert the tube 5 times (do not shake the tube).
Observed your tube. Any changes?
7. Add 5 drops of protease to your tube.
8. Place the cap on your tube and gently invert it a few times.
9. Place your tube in a test tube rack or beaker in the water bath and incubate at 50oC for
10 minutes.
10. Holding your tube at a 45o angle, fill you tube with cold alcohol by adding
approximately 10ml to your tube. It will take repeated additions to add 10ml using the
disposable transfer pipet.
11. Place your cap on your tube and let it site undisturbed for 5 minutes. Observe and
note any changes in your preparation.
12. After 5 minutes, slowly invert the tube 5 times to help the DNA, which has begun to
precipitate, to aggregate.
Questions
1. What is the purpose of the homogenization buffer? Describe the role of the different
chemicals.
2. Describe the appearance of the DNA precipitate from step 12.