Bi 6 Biotechnology for Everyone, Laboratory

Exercise 9. DNA Isolation

Name:___________________________________________ Date: _______________________

Group No.: _______________________________________ Instructor: ___________________

Materials:
5 mL E. coli (grown in LB; 18-24 h)
Sterile empty screw cap test tube
Thermometer

Test tube holder
Pot holder

Hot water bath

Sterile blue tips (wide ends)

Cold 95% ethanol

Light-colored (colorless) dishwashing liquid or shampoo or detergent (not antibacterial)
Test tube rack

Sterile pasteur pipettes

Microcentrifuge tubes

Procedure:

1. Transfer 1 mL detergent/dishwashing liquid/shampoo in the sterile empty test tube. Pour the E.
coli broth culture into the tube of detergent. Cap the tube and invert it slowly several times to
allow mixing of the solution without foaming. Place the tube in a hot water bath (55-60°C) for
10 minutes. Observe changes in the clarity of the solution.
2. Remove the tube from the hot water bath and transfer to a test tube rack. Add cold ethanol to
the test tube (fill a Pasteur pipette with ethanol and put it to the bottom of the test tube before
releasing the ethanol into the solution) to create an alcohol layer on top of about 1 cm. For best
results, the ethanol should be as cold as possible.
3. Let the solution sit for 2 - 3 minutes. Refrain from disturbing the solution and shaking the test
tube. Watch the white DNA precipitate out into the alcohol layer (DNA has the appearance of
white mucus). Transfer the DNA precipitate to sterile microcentrifuge tubes using the blue tips
with wide ends. Store the DNA samples in the freezer (label with type of sample, section and
group number).

Department of Biology, School of Science and Engineering

Ateneo de Manila University
Bi 6 Biotechnology for Everyone, Laboratory
Guide Questions:
1. What is a DNA? What is its structure? What is its significance?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________

2. Describe the appearance of the DNA you were able to isolate. Do you expect your extracts to be
pure DNA isolation? What contaminants do you expect?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________

3. How can you prove that what you were able to isolate was DNA?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________

4. Enumerate other good sources for extracting DNA.

________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________

5. What is the use/purpose of detergent in DNA isolation?

________________________________________________________________________________
________________________________________________________________________________

6. What is the use/purpose of cold alcohol in DNA isolation?

________________________________________________________________________________
________________________________________________________________________________

Department of Biology, School of Science and Engineering

Ateneo de Manila University