TOTAL DNA EXTRACTION AND QUANTIFICATION

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 3 different samples including porcine meat
as positive control. Cut and mince 2 mm2 fragment of each food sample
using separate blade. Then, carefully transfer each sample into a 0.2ml PCR
tube.
Lysis reaction
1. Combine the following in each PCR tube containing sample:
Reaction setup
Volume (l)
10X KAPA Express Extract Buffer
10
1 U/l KAPA Express Extract Enzyme
2.0 (2 U)
PCR-grade water
Up to 100
2. Vortex to mix the reaction.

3. Incubate for 10 minutes at 75C.

During this step, cells are lysed, nucleases and proteins degraded and
DNA released.
4. Incubate for 5 minutes at 95C to inactivate the thermostable KAPA
Express Extract protease.
5. Vortex reaction product for 2-3 seconds. Centrifuge at maximum speed
for 1 minute to pellet debris.
6. Transfer DNA-containing supernatant to a fresh tube.
7. The DNA sample is ready to be analyzed for its quality. Use 5l of DNA
extract for the 0.8% agarose gel electrophoresis. DNA extracts are readily
to be used in PCR and can be stored in TE buffer at -20C for long term
storage.
Note: 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
containing 1X TAE buffer.
2. Mix 5l of DNA sample with 1l 6X loading dye.
3. Mix 2l DNA-HindIII marker with 2l loading dye.
4. Load the sample mixture and DNA marker into the wells.
5. Apply a voltage of 75 volts for 75 minutes.

B. DNA Quantification & Qualification

Spectrophotometric determination of DNA concentration
Experiment Procedures
1. Dilute 3 l of DNA with 297l 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.