Universiti Tunku Abdul Rahman

Faculty Lee Kong Chian Faculty of Engineering and

Science
Department: Department of Mechatronics and BioMedical
Engineering
Course Code and Name UEMB2223 Biochemistry and Molecular Biology
Experiment No.: 1/5
Title of Experiment: Extraction and Characterization of
Chromosomal DNA
Laboratory Room No. and Name: KB615
Experiment Duration (hour): 3 hours
Number of Student per Group 4 students x 4 groups
Number of Student per Session 16

Equipment and Materials

Quantity estimation
*Item
Item Description (e.g. per set/group of
category
student)
Escherichia coli cell culture SP
Lysis buffer CH
Phenol-chloroform-isoamyl alchohol CH
Potassium acetate CH
Ethanol CH
Sodium dodecyl sulfate (SDS) CH
TE buffer CH
Standard λ DNA [525 g/ml] SP 100 ng
Ice C
Microcentrifuge tubes C
Water bath E 1 unit
Quartz cuvettes W
Spectrophotometer for UV measurements E 1 unit
Table-top centrifuge E 1 unit
Micropipettes and tips C 2 sets
Test Tube Vortex Mixer E 1

*Item category
SP Sample or specimen
C Consumable
CH Chemical
W Labware, glassware, tool, and
components
E Equipment
S Software
1 Latest updated: 26 Feb 2018
Procedure
PART A: DNA Extraction
1. Add 1ml of an overnight E.coli culture to a 1.5ml microfuge tube.
2. Centrifuge at 12,000 rpm for 2 min.
3. Remove the supernatant.
4. Resuspend the cell pellet in 600 μl of Lysis Solution (LS) and then incubate at 80°C
for 5 min to lyse the cells.
5. Cool the tube contents to room temperature.
6. Add 600 μl of phenol-chloroform-isoamyl alchohol (PCI) [use bottom layer only as the
top layer is TRIS] to the cell lysate.
7. Vortex for 20 seconds to mix.
8. Centrifuge at 12000 rpm for 3 min.
9. Transfer the supernatant containing the DNA to a clean 1.5ml microfuge tube
containing 600 μl of room temperature absolute ethanol and 300 μl potassium acetate.
10. Gently mix by inversion.
11. Centrifuge at 12000 rpm for 2 min.
12. Carefully pour off the supernatant and drain the tube on clean absorbent paper.
13. Add 600μl of room temperature 70% ethanol and gently invert the tube several times
to wash the DNA pellet.
14. Centrifuge at 12000 rpm for 2 min.
15. Carefully pour off the ethanol and drain the tube on clean absorbent paper.
16. Allow the pellet to air-dry for 10–15 min.
17. Add 100 μl of TE buffer to the tube and rehydrate the DNA by incubating at 65°C for
1 hr.

PART B: Ultraviolet Measurement and Denaturation of Isolated DNA

1. Transfer 100 μl of the extracted DNA (from Part A) to a quartz cuvette.
2. Blank the reading first with TE buffer before measuring the absorbance.
3. Determine and record the A260 and A280 on the DNA sample. (If the A260 is greater than
1, quantitatively dilute the sample until the absorbance reading is between 0.5 and 1.0).
4. Pipett 5μl of standard λ DNA (525 μg/mL) into each of three microcentrifuge tubes.
5. Maintain one tube at room temperature and place the other two in a 90°C water bath
for 15 min.
6. After the incubation, remove the tubes. Determine and record the A260 and A280 on
these three DNA standards.
7. Quick-cool one heated tube in an ice bath and allow the other heated tube to cool slowly
to room temperature over a period of about 1 hr.
8. After 1 hour, measure and record final A260 and A280 readings on the three tubes.
9. Calculate the A260 /A280 ratio for both extracted DNA and standard λ DNA.
10. Calculate the A260(T) /A260(25°C) ratio for each of the three tubes.

2 Latest updated: 26 Feb 2018