ISOLATION OF TOTAL DNA FROM EPITHELIAL CELLS
Principe:
First hundreds to thousands of epithelial cells are isolated in the form of suspension by
simple washing bucal mucous membrane with isotonic solution of NaCl. Subsequently, part
of cells is isolated by centrifugation from an aliquot of suspension. These cells are
resuspended in small quantity of isotonic solution of NaCl that leads to their higher
concentration. Chelex® - polystyrene “beads” with covalently immobilized imid-acetic acid
(IDA) - is added to this suspension. In following steps, IDA binds up bivalent metal ions and
by this way protects DNA molecules against DNases enclosed particularly in saliva. DNase
activity, which digests DNA into short fragments, is strongly dependent on the presence of
bivalent metal cations particularly Mg2+. The suspension is subsequently incubated at 99 °C
that leads not only to disintegration of cells and release of DNA into solution, but also to heat
denaturation and precipitation of the majority of cell proteins. Precipitate and Chelex® are
subsequently removed from the cell lysate by centrifugation and chromosomal DNA is then
present in the supernatant. DNA, obtained by this way, is partly contaminated by cell RNA
and thermo stable proteins and peptides; nevertheless the quality of preparation is sufficient
for following use in PCR.
Material and chemicals:

pots with 10 ml solution of sterile 0.9% of NaCl

1.5 ml micro tubes with storage solution of sterile 0,9% NaCl

Chelex® (Sigma) - 10% suspension in water

1.5 ml micro tubes („eppendorfs“)

automatic pipettes (P-20, P-200, P-1000)

laboratory centrifuge for 1.5 ml micro tubes

thermo-block

spectrophotometer for DNA measurement

protective gloves
Performance:
(!) Work in protective gloves! During pipeting always use a new tip – by this way protect
your samples against contamination of DNA from another participant. Throw used plastic
aids into designated trash dishes.
1.
Take into your mouth 10 ml of 0.9% water solution of
NaCl and intensively turn it over and with help of teeth
scratch
lightly
bucal
mucous
membrane
for
approximately 1 min.
2.
Bring the suspension of epithelial cells, which has arisen
by this way, into a pot and mix the content.
3.
Pipette 1 ml of the suspension (pipette P-1000) into an
advance signed 1.5 ml micro tube.
4.
Separate cells by centrifugation at 3 500 × g for 4 min
(samples have to be arranged symmetrically in a
centrifuge rotor = balanced rotor).
5.
Take out a micro tube from the rotor and make sure, if
there is formed a cell pellet at the bottom. With help of a
pipette P-1000 remove carefully a supernatant back into
the pot (The pellet is not attached too tightly on the wall! The small
quantity of liquid can stay in the tube.).
6.
Resuspend the pellet intensively - rack a closed micro tube
on a micro tube stand.
7.
Pipette 200 µl of 10% Chelex ( pipette P-1000) into micro
tube with your sample (be sure that you pipette the
polystyrene „beads“ – they are important!! see part
Principle , Chelex can be shaken before use).
8.
Put the 1.5 ml micro tube into a thermo-block and
incubate it at 99 °C for 10 minutes. Cell disintegration by
heating
(CELL
LYSIS)
is
in
progress
with
precipitation
(DENATURATION) of the majority of PROTEINS.
9.
Pipette the content of ml micro tube (pipette P-200) into a
new advance signed 1.5 ml micro tube and centrifuge it at
10 000 × g for 2 min. CELL RESTS and PRECIPITATED
PROTEINS of cells and intercellular matter are REMOVED.
10.
Pipette 60 µl of the supernatant containing genomic DNA
(pipette P-200) into a new advance signed 1.5 ml micro
tube (it must be without Chelex particles!). Store DNA
for short time at 4 ºC (in refrigerator) or for long time at 20 ºC (in freezer).
11.
DNA concentration is measured by spectrophotometer
(Nano Photometer IMPLEN).