DNA EXTRACTION FROM BACTERIA

Prepared by the Office of Biotechnology, Iowa State University

TEACHER PREPARATION AND INSTRUCTION GUIDE

Preparation for the DNA extraction experiment should begin at least 36 hours in advance of the laboratory
period.

The supplies can best be provided to the class in groups of four students. A test tube rack works well to
provide each group of four students with the following:

* 4 capped test tubes containing E. coli, one for each student. Each tube should contain about 5 ml of
the E. coli solution or be half full, whichever is less. The tubes should be labeled "E. coli".
* 4 glass rods or similar device for spooling DNA, one for each student.
* 4 capped test tubes containing light-colored dishwashing liquid or shampoo, such as Dawn or Suave
Daily Clarifying Shampoo, one for each student. The tubes should be labeled "Detergent" . The
amount of detergent in each tube should be about 1/5 the quantity of the E. coli solution in the tubes
described above ie. 1 ml of detergent if 5 ml of the E. coli solution are in the tube.
* 1 capped test tube full of 95% ethanol, to be shared by the students. The tube should be labeled
"Ethanol".
* 1 Sharpie marking pen, to be shared by the students.
* (Optional) 4 toothpicks, one for each student, if meat tenderizer is used.

The group of four students also will need:

* 1 pasteur pipette or medicine dropper with a rubber bulb attached.

* 4 copies of the laboratory instructions, one for each student.
* (Optional) 1 container with enough meat tenderizer to obtain 8 toothpicks full, to be shared by the
students. The container should be labeled "Tenderizer".

The teacher should have available for the entire class:

* 1 bottle of E. coli with sufficient volume to prepare the test tubes for the students before the class
begins.
* 1 hot plate with a range of adjustable temperatures and 1 square baking pan large enough to hold the
test tubes racks described below. The hot plate and pan will be used to create a hot water bath of 55-
o
60 C.
* 1 thermometer.
* Enough test tube racks to hold the tubes of the students while they are incubated in the hot water
bath.
* 1 Sharpie marking pencil
DNA EXTRACTION FROM BACTERIA
PAGE 2

PREPARATION OF THE E. COLI SOLUTION

Step 1. Preparation of the Luria broth (LB).

There are two methods for preparing the LB. One method utilizes a premix that contains all the necessary
nutrients. The second method is to prepare the necessary nutrient mixture from the individual ingredients.

Method when the LB premix is used: The volume of distilled water to use is determined by multiplying
the volume of the E. coli solution that will be added to each test tube for the students X the number of students. It
is best to use some extra water to account for possible spillage and other factors. Weigh 25 mg LB premix/ml of
distilled water into a bottle. (For a 1,000 ml volume of water, add 25 g of the LB premix.)

Method when the LB is made from separate ingredients: Weigh 5 mg of yeast extract/ml of distilled water
and place in a bottle large enough to hold the desired volume of water. Weigh 10 mg of NaCl/ml of water and add
to the bottle. Weigh 10 mg of Tryptone/ml of water and add to the bottle. Add the desired volume of distilled
water to the bottle. (For a 1,000 ml volume of LB, use 5 g of yeast extract, 10 g of NaCl, 10 g of Tryptone, and
1,000 ml of distilled water).

The bottle containing the LB should not be more than half full. Sterilize the LB by one of the following
methods:
a) Autoclave the bottle for 15 minutes.
b) Place it in a pressure cooker at 15 pounds for 15 minutes.
c) Place it in boiling water for 30 minutes.
The cap on the bottle should be loose when it is sterilized to permit hot vapor to escape. After sterilization is
complete, allow the broth to cool until the bottle is comfortable to hold, then close the cap tight. Store in the
refrigerator until it is used in Step 2.

Step 2. Preparation of the E. coli solution

The sterile LB prepared in step 1 should be brought to room temperature. E. coli should be added to the
LB in a manner that avoids contamination from other organisms. There are two methods that can be used to
inoculate the LB with the E. coli.

One method involves the use of a sterilized inoculation loop or a sterilized paper clip bent into a loop to
transfer bacteria. The inoculation loop or paper clip is sterilized with a bunsen burner and cooled by touching the
loop to a blank section of agar. A colony or section of E. coli the size of this O is put on the loop without
removing the agar. The loop is inserted into the LB and the cells are removed from the loop by stirring for a few
o
seconds. The bottle of broth is capped loosely and incubated at 37 C.
DNA EXTRACTION FROM BACTERIA
PAGE 3

The second method involves the use of a sterile toothpick, a sterile tweezer, and a culture of E. coli. The
toothpick is sterilized by wrapping it in aluminum foil and placing it in an autoclave for 15 minutes, a pressure
o
cooker at 15 pounds for 15 minutes, or a kitchen oven at 350 F for 15 minutes. A match is used to flame the end
of a pair of tweezers. The sterile toothpick is picked up with the sterile tweezer, a colony or section of E. coli the
size of the O is put on the toothpick, and dropped into the bottle of LB. The bottle is capped loosely and incubated
o
at 37 C.
o
The LB is incubated at 37 C with frequent shaking, if possible, for at least 24 hours and preferably 36 or
o
more hours. The temperature should not exceed 40 C to avoid killing the bacteria. The solution is dispensed into
individual test tubes for the students just before the laboratory. It is not necessary to use sterile procedures when
filling the tubes.

SETTING UP ON THE DAY OF THE LABORATORY

The supplies for each group of four students should be placed on the laboratory bench. The hot water bath
o
at 55-60 C is set up by placing the square pan on the hot plate, placing the test tube rack(s) in the pan, adding
water to within about 1 inch of the top of the pan, and turning on the hot plate to the appropriate setting. It is best
to have identified in advance of the laboratory day the approximate setting on the hot plate dial that will provide the
desired temperature.

COMMENTS ON THE STUDENT INSTRUCTIONS

Step 4. It is possible to mix the ethanol and water to get more DNA to precipitate. Add enough additional ethanol
to form a 2 cm layer above the water. Cap the tube and mix it thoroughly by shaking. Allow the tube to stand for
10-15 minutes, or until the next laboratory, if necessary. A large amount of DNA should appear as a white mucus-
like substance in the tube.

CLEAN-UP AFTER THE LABORATORY

E. coli solution that was not dispensed can be autoclaved for 15 minutes or boiled 30 minutes, then poured
down the drain.

The isolated DNA in the test tubes can be poured down the drain. The test tubes, pipettes (glass part), and
glass rods or other device used for spooling can be washed and saved for future use.

Unused meat tenderizer can be poured back in the bottle from which it was obtained and saved for future
use. The toothpicks can be put in the regular trash.

revised 3/94
DNA EXTRACTION FROM BACTERIA

STUDENT INSTRUCTIONS

DNA carries in its molecular structure the genetic information for cell development and behavior.
Consequently, all living cells contain DNA. Scientists can isolate DNA from cells of any plant, animal, or
microorganism. In this laboratory procedure, you will isolate DNA from E. coli bacteria.

Step 1. Write your name or initials on a test tube containing detergent. Pour the E. coli solution from a test
tube into your tube of detergent. Cap the tube, invert it slowly several times to mix the solution
o
without foaming, and place the tube in a hot water bath at 55-60 C for 10 minutes. Observe changes in
o
the clarity of the solution. Detergent and heating at 55-60 C dissolve the fats in the cell walls of the
bacteria, which frees the DNA.

Step 2. (Optional) Remove the tube from the hot water bath. Add two flat toothpicks full of meat tenderizer to
o
the tube, cap the tube, mix gently but thoroughly, and return the tube to the 55-60 C water bath for 20
minutes. (Meat tenderizer contains papain, an enzyme to break down any proteins that may be attached
to the DNA.)

Step 3. Remove the tube from the hot water bath. Add cold alcohol to the test tube to create an alcohol layer on
top of about 1 cm. For best results, the alcohol should be as cold as possible. The alcohol can be added
to the solution in at least three ways. (a) Fill a pasteur pipette with alcohol, put it to bottom of the test
tube, and release the alcohol. (b) Put about 1 cm of alcohol into the bottom of a test tube and add the
solution. (c) Slowly pour the alcohol down the inside of the test tube with a pasteur pipette or
medicine dropper. DNA is not soluble in alcohol. When alcohol is added to the mixture, all the
components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the
alcohol layer.

Step 4. Let the solution sit for 2-3 minutes without disturbing it. It is important not to shake the test tube.
You can watch the white DNA precipitate out into the alcohol layer. When good results are obtained,
there will be enough DNA to spool on to a glass rod, a pasteur pipette that has been heated at the tip to
form a hook, or similar device. DNA has the appearance of white mucus.

Prepared by the Office of Biotechnology, Iowa State University. revised 3/94