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Introduction
DNA isolationis a process of purification of DNA
from sample using a combination of physical and
chemical methods.
The first isolation of DNA was done in 1869 by
Friedrich Miescher.
Falcon tube
Eppendorf tube
Centrifuge
vortex
Protocol
(1)Firstly wash your mouth gently with water in
order to remove any foreign particles.
(2)Move a new toothbrush over the inner surface
of the cheeks scraping the cells lining the mouth
cavity.
(3)Use 2 ml of phosphate buffer saline (PBS) to
rinse the
mouth.
(4)Collect the (PBS) with cells in a 15ml falcon
tube.
(5)Centrifuge the sample at high speed(3000rpm)
for 10min to collect the pellet.
(6)Add 2 ml lysis buffer 10l proteinase k and 1
ml guanidine chloride and 300l NH4 acetate
incubate at 37C for 15min.
During the extraction of DNA (or nucleic acids in
general), there is a lot of contaminating proteins
present. These contaminants must be removed.
Proteinase K, which is a broad spectrum serine.
Ammonium acetae use to percipitate DNA from
(7) Cool to room temperature and then add 2ml
per chilled chloroform (extract proteins and lipids
away from the DNA).
DNA
Protein
layer
Chloroform
(8)Vortex and then centrifuge for 5min at
3000rpm.
(9)Collect the upper layer to a new tube and add
10ml cold absolute ethanol shake and keep at
20C for at least 2hrs or overnight (chilled
absolute ethanol precipitates the DNA).
(10)Centrifuge at 3000rpm from15-20min
carefully drain the supernatant invert the tube on
a tissue paper for 5min.
(11)Wash the pellet with 4ml of 70%EtoH
(rehydrate wash the pellet and remove any salt
that precipitated with DNA).
(12)Centrifuge at 3000rpm for15min.
(13)Pour off the supernatant and allow the pellet
to dry for1-2hrs(avoid over dry).
(14)Re-suspend the pellet in 200l d H2O or
TE(Tris-EDTA) briefly vortex and put in 4C
overnight.
(15)Aliquot the DNA into stock solution ( store at-
20C) and working solution(store at 4C).
(B)DNA extraction from
(1)Phenol
Bloodchloroform
Protocol
(1)Collect 3-5ml blood in EDTA tubes.
(2)Add 10 ml of Red Cell Lysis Buffer (RCLB),mix
well and centrifuge for 5 min at 6000rpm.
(3)Repeat the previous step until a clear pellet of
white blood cells appears at the bottom of the
tube.
(4)Discard the supernatant and add 800 l of
White Blood Cell Lysis Buffer (WCLB) and 10 l of
proteinase K (10mg/ml) to the precipitate and
incubate overnight at 37C or at 65Cfor 2hrs.
(5)Add equal volume of
phenol/chloroform/isoamyl alcohol, mix and
centrifuge at 6000 rpm for 5 min.
(6)Transfer the upper layer in a clean tube, add
equal volume of chloroform /isoamyl alcohol,
vortex and centrifuge for 5 min at 6000rpm.
(7)Transfer the upper layer to a clean tube, add 2
volume of 95% cold ethanol and then add 1:10 of
sample volume of 3 M Na acetate and incubate at
-20C overnight or 2hrs.
(8)Centrifuge for 10 min. at 12000 rpm and
discard the supernatant.
(9)Add 2 ml of 70% ethanol, mix well and
centrifuge for 7 min at 12000 rpm, then discard
the supernatant.
(10)Repeat: the previous step.
(11)Discard the supernatant and allow the pellet
to dry for 15 min.
(12)Add 100l of TE buffer or ddH2O and store at
20C .
(2)Protein
(1)Take 300l of WBCs in precipitation
an eppendorf tube; add
100l of protein precipitation solution.
(2) Mix by vortexing gently; incubate for 5 min in
ice.
(3) Microfuge at full speed for 6 min.
(4)Collect the liquid supernatant in a new
eppendorf tube, discard the pellet .
(5)Add 300ul of isopropanol, mix gently for 5 min,
spin for 5 min in micrfuge .
(6)Pour off the supernatant without disturbing the
pre
cipitate.
(7)Wash with 70% ethanol (600l).
(8)Spin for 3-5 min.
(9)Pour off the supernatant without disturbing the
(10)Dry the pellet at room temperature.
(11) Re-suspend the pellet in 100 l TE buffer
overnight to dissolve.