NAME: ________________________________
TOTAL DNA EXTRACTION BY FREEZE-THAW
PURPOSE
To extract total DNA from an unknown microorganism, utilizing freezethaw as the cell lysis method. To prepare DNA samples as a template for
amplification by Polymerase Chain Reaction (PCR) for further genetic
analyses.
INTRODUCTION
All cells, the basic structural and functional unit of life, consist of living
material bounded by layers of membranes made of lipids, proteins, and
some other compounds. Cell lysis is the first step in the process of DNA
purification. The DNA genome contains all the genetic information of an
organism, and is protected from the external environment by the cell
membrane. In order to release the genetic material for study and
analysis, cells must be broken open, or lysed. There are several methods
available for cell disruption including physical and chemical techniques.
For chemical disruption, different compounds are used to dissolve and or
disrupt cell membranes. For example enzymes such as lysozyme and
proteinase K, detergents such as SDS and CTAB, and some other strong
chemicals will allow the DNA to be released. Physical techniques are
often the first method of choice for cell disruption, and include
mechanical disruption, liquid homogenization, sonication, freeze-thaw,
and manual grinding. For this laboratory exercise, freeze-thaw will be
used because it is a very common method used to lyse bacterial cells.
This technique involves quickly freezing a cell suspension in a dry iceethanol bath and then quickly thawing the cells in a very hot water bath
(between 80C to 90C). This method of lysis causes the cell to swell and
shrink, ultimately breaking due to ice crystals formed during the freezing
process. Several freeze-thaw cycles are needed to facilitate the cell
membrane breakage and the release of DNA for further experimentation.
Now let’s try to extract DNA!
MATERIALS
Latex gloves
Dry ice
Ethanol (alcohol)
Beaker
Goggles
Unknown culture
Microcentrifuge tubes rack
Sterile distilled water
Plastic container
Hot plate or candle warmer
Micropipette tips
Hot water
Microcentrifuge tubes
Microcentrifuge
Permanent marker
Micropipettes
PROCEDURE
1. Before beginning be sure to glove your hands in order to prevent
contamination. Put on safety glasses.
2. In a beaker or a glass container put some tap water to heat on top of
the hot plate or a candle warmer. The water does not have to be
boiling, but should be 80C to 90C.
3. While the water is heating up, take the plastic container and put
some dry ice in it along with enough ethanol to create a slurry. Make
sure that the dry ice is covered with the liquid and that there is space
to place the microcentrifuge tubes in the slurry.
Caution: Dry ice is cold enough to burn your skin. When
handling dry ice, use the kitchen mitts provided. Be sure to
keep wearing your eye protection while working with the hot
and cold baths.
4. Take the tube with your culture that contains the unidentified
bacteria grown on a specific media and place it the microcentrifuge
with the purpose to pellet the cells and remove the liquid culture
media. Centrifuge the sample for five minutes at the maximum speed.
Before you centrifuge your sample make sure that the
microcentrifuge has been balanced by placing another tube with
the same volume of liquid as your sample tube on the opposite
side of the rotor. The microcentrifuge rotor must have an even
number of tubes placed one across from the other to create
balance.
5. After you have centrifuged your unknown sample discard the
supernatant (the liquid that is in the top of the sample tube) by
dumping it in an empty container to be discarded properly later.
Gently tap the tube on a paper towel to get rid of as much
supernatant as possible. Take care not to disturb or dislodge the
pellet on the bottom of the tube.
6. After the supernatant has been discarded, resuspend the cell pellet in
500 L of sterile distilled water. Using the 1000 L micropipette,
dispense 500 L of the water into your sample tube, close the tube,
and mix until you get a homogenized solution.
7. Place the closed tube in the dry ice-ethanol bath using forceps to hold
the tube. Keep the tube in the bath for three minutes to freeze. Then
quickly place the tube in the hot water bath for three minutes. This
process is known as a freeze-thaw cycle. Repeat the same process for
a total of three cycles. Make sure that you mix your sample tube
between each cycle.
8. After you have completed the three freeze-thaw cycles, proceed to
centrifuge your sample tube for five minutes at maximum speed in the
microcentrifuge. Again DO NOT FORGET TO BALANCE THE
MICROCENTRIFUGE.
9. At this point, be careful not to lose the supernatant. We want to save
the supernatant that contains the DNA and discard the pellet that
contains cellular debris. Using a micropipette, transfer the
supernatant into a new clean, sterile microcentrifuge tube.
Now, you are ready to amplify your DNA millions and zillions of times by
using the Polymerase Chain Reaction method!
QUESTIONS AND CONCLUSIONS
1. Name three other techniques that can be used to lyse cells and
release their DNA.
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2. Explain briefly the freeze-thaw method.
3. What do you have to do every time you are ready to centrifuge a
sample?
4. What does PCR stand for?