LAB 1: TOTAL DNA EXTRACTION AND

QUANTIFICATION
The objectives of this lab are:

To isolate genomic DNA from selected commercially processed food

products using KAPA Kit Extract Extraction Kit (KAPA Biosystem).

To determine the quality and quantity of the isolated DNA via agarose gel
electrophoresis and spectrophotometric method

To prepare isolated DNA to be used as DNA template for PCR lab session.

A.

DNA Extraction

Introduction
KAPA Express Extract is a novel thermostable protease and buffer system
that allows for the extraction of PCR-ready DNA from various tissue types in
as little as 10 minute. The KAPA Express Extract system has been designed
for optimal tissue lysis and sample preservation. Unlike existing protocols
that rely on proteinase K digestion, KAPA Express Extractions are
conveniently performed in a single-tube, without the need for hazardous
chemicals and multiple washing steps. This greatly reduces the risk for
sample loss and contamination.
Methodology
Starting material
Each group is provided with commercially processed food products. Cut and
mince 2mm2 fragment of each food sample using separate blade. Then,
carefully transfer each sample into a 1.5ml microcentrifuge tube.
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Lysis reaction
1. Combine the following in each 1.5ml tubes which already contained food
sample:
Component
10X KAPA Express Extract Buffer
1 U/l KAPA Express Extract Enzyme
PCR-grade water

Volume (l)
10
2.0 (2 U)
Up to 100

2. Pulverize the sample with the other components using plastic mortar.
3. Vortex to mix the reaction.
4. Incubate for 10 minutes at 75C. During this step, cells are lysed,
nucleases and proteins degraded and DNA released.
5. Incubate for 5 minutes at 95C to inactivate the thermostable KAPA
Express Extract protease.
6. Vortex reaction product for 2-3 seconds. Centrifuge at maximum speed
for 1 minute to pellet debris.
7. Transfer as much as possible supernatant which contains DNA into a fresh
1.5ml tube.
8. For long term storage, store the tubes at -20C.
Notes obtained from the KAPA Express Extract kit website:
Quantification of DNA extracts is not recommended. Crude DNA extracts are
likely to contain cellular contaminant that will affect the absorbance of the
sample in the range of 260-280nm and result in inaccurate DNA
concentration determinations based on spectrophotometric methods.
One percent Agarose Gel Electrophoresis
1. Prepare 1% agarose gel and place the gel into the electrophoresis tank.
The wells of the gel should be located near to the negative electrode.
2. Submerge the gel with 1X TBE buffer.
3. Mix 5l of DNA sample with 1l 6X loading dye. Then load this mixture
into any lane on the gel from lane 2-7.
4. Mix 2l DNA-HindIII marker with 2l loading dye (If the marker is a
premixed version, just directly load 3l). Load DNA marker in both lane 1
and 2 of the gel.
5. Close the electrophoresis tank using its lid. Apply a voltage of 75 volts for
75 minutes.
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6. After the run, transfer the gel in staining solution for 10 min, followed by
into destaining solution for 10 minutes.
7. Observe the gel under UV using the Biorad Image Analyser.

B. DNA Quantification & Qualification

Spectrophotometric analysis

1. Dilute 3 l of DNA with 597l deionised water and read at A 230, A260 and

A280. The A260/A280 ratio provides an estimate of the purity of the DNA. In
a pure sample, this ratio is approximately 1.8. Lower values indicate
protein or phenol contamination. A230 should be less than A260 and may
be the same as A280. High A230 reading indicates that residual phenol
remains in the preparation. An A260 of 1 corresponds to approximately
50 l/ml of double-stranded DNA in a 1 cm quartz cuvette. Nucleic acid
concentration is calculated as follows:

[DNA] = A260 X 50 X dilution factor (dilution factor)

(ng/ l)
Trouble shooting

DNA purity = A260 /A280

Table 1: Common problems in DNA extraction and appropriate

solutions.