This is a modification of a salting
out procedure as described by Miller et al., (1988), evaluated at the DNA
Laboratory, Medical School, Malta. When analysed by spectrophotometry >95%
of extracted genomic DNA gave a purity ratio of DNA to proteins in the
range of 1.7 - 1.9 and a concentration of 100ng/µl.
In the following protocol DNA was
extracted from peripheral blood leucocytes using 3mls of whole blood. It
was observed that on prolonged storage of whole blood at -20 and -80ºC
, DNA yield decreased considerably due to degeneration of the
white blood cells. Volumes of buffers and reagents used may be adjusted
according to volume of whole blood used.
blood was collected in di-potassium EDTA containers (never use heparin
3mls of whole blood were transferred to a sterile conical
centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing
buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2
EDTA; pH 7.4) were added.
solution was left for 10 minutes at RT with occasional mixing by
inversion followed by centrifugation for 5 minutes at 4000 rpm.
centrifugation the supernatant was discarded and a white pellet was observed at the bottom of the tube. This pellet
was washed for at
least 3 times by adding 3mls of erythrocyte lysing buffer and repeating
steps 3 and 4. It is important to breakdown the pellet and rinse it well
in erythrocyte lysing buffer in order to clean the white blood cells
from remaining heme.
1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0)
of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), were added to
the pellet. The tubes were incubated at a temperature of 37 - 55ºC
(optimal temp for Proteinase K activity) overnight in a water bath or
incubator. During this step the white blood cells' membranes are
denatured and DNA goes out in solution.
incubation, 1.5mls of SE buffer together with 750 ul
of 6M (saturated) NaCl were added to each tube, followed by the addition
of 3.75mls chloroform.
tubes were mixed vigorously (on a vortex) for about 20 sec with
occasional mixing for at least 30min. Alternatively you can leave the
tubes on a rotator for 1 hour.
emulsion was centrifuged for 10 minutes at 2000 rpm with
minimal breaking force. After centrifugation 2 phases were observed
(important not to disturb the interphase). During this step DNA
is extracted into the supernatant and proteins separated into the lower
upper aqueous phase (containing the DNA) was transferred into a clean and
sterile conical centrifuge tube using a sterile Pasteur pipette,
followed by the addition of an equal volume of isopropanol (double the
volume of 100% ethanol can also be used)
was precipitated by gentle swirling of the tube and was observed
visually as a white thread like strand.
a sterile spatula or loop the DNA was transferred a sterile microcentrifuge tube containing 1ml of 75% ethanol.
DNA was washed by inversion to clean it from any remaining salts and
the tube centrifuged at 11000g for 4 minutes. The supernatant was
discarded taking care not to discard the pellet. This step was repeated once
discarding the supernatant the pellet was dried from excess ethanol
either by leaving the tubes open and
inverted in an oven at around 50 - 65ºC for an hour.
The dried pellet was resuspended in 300µl TE buffer (1M Tris-HCl; 0.5M EDTA; pH
8.0) and left overnight on a rotator.
concentration was determined either by agarose gel electrophoresis or
spectrophotometry and adjusted to the desired concentration by adding
more TE buffer. It is important to note that before adjusting and
reading DNA concentrations one must obtain a homogeneous sample of DNA
which is not quite easy acquired since DNA is very viscous. To adjust to
lower concentration one must use other quantitation methods such as
Picogreen (Molecular Probes)
using spectrofluorometry as spectrophotometry is not accurate and
sensitive at very low concentrations.
Fig. 1. Col1a1 Sp1
Fragment amplified by PCR using DNA extracted by the above method.
For routine storage the best condition is at +4 degrees
Celsius in TE buffer (pH 8.0). Highly purified DNA can be stored for
Dried DNA pellets can be stored at -20 degrees Celsius
for up to 6 months.
DNA precipitated in ethanol can be stored indefinitely
at -20 degrees Celsius. For long term storage -80 degrees Celsius is
DNA EXTRACTION FROM CHEEK CELLS
Miller SA, Dykes DD, Polesky HF 1988 A
simple salting out procedure for extracting DNA from human nucleated
cells. Nucleic Acids Res
Green et al. 1998 Genome Analysis: A Laboratory Manual. Cold Spring Harbor