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DNA Extraction

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.

He was the first researcher to isolate and


identify nucleic acid.
Principle
Detergent-based lysis arises from incorporation
of detergent into the cell membrane solubilizing
lipids and proteins in the membrane creating
pores within the membrane and eventually full
cell lysis.
Samples
Amniocytes or
Whole blood amniotic fluid
Buffy coat Dried blood spots
Serum or plasma Fresh or frozen
Bone material tissue (biopsy
Buccal cells material)
Cultured cells Sputum, urine,
CSF, or other body
fluids
Fixed or paraffin-
embedded tissue
(A)DNA extraction from
Buccal cavity
Guanidine chloride
Required
(1)Phosphate buffered saline (PBS)
Preparation
1.Dissolve one tablet in 100ml dH2O(Or follow the
instruction on the bottle)
2.Autoclave for 10 minutes.
(2)Lysis Buffer
The lysis buffer contains the detergent sodium
dodecyl sulfate SDS(to break apart the
membranes)salts and sugar (to increase the
osmotic pressure outside of the cell and break
open the cells) and the buffer tris (to maintain the
Preparation
PH of the solution).
(I)5 ml1M Tris pH 8.0
(II)10 ml 0.5M EDTA pH 8.0
(III)5 ml1M Sucrose.
(IV)5 ml 10% SDS.
(V)2 ml 5M Na Cl. 1.Add distilled water to bring
volume to 100 ml.
2.Mix well.
Lysis buffer needs to be made up fresh each time
you do this lab because bacteria will start to grow
(the sucrose provides nourishment).
Check for contamination by swirling the
container and looking for floating particles or
bacterial threads.
(3)10% SDS (Sodium Lauryl Sulfate)
Sodium lauryl sulfate (SLS) or sodium dodecyl
sulfate (SDS or NaDS) (C12H25SO4Na) is an
anionic surfactant (Denaturing detergents)
disrupt membranes and denature proteins by
breaking protein:protein interaction.
Preparation
Makes 100 ml.
Store at room temperature (indefinitely).
Avoid inhaling SDS powder; wear a mask over
mouth & nose.
1.Dissolve 10 g electrophoresis-grade SDS (m.w.
288.37) in 80 ml deionized water.
2.Add deionized or distilled water to make 100 ml
(4)1 M Sucrose
(I)8.55 g sucrose
(II)Distilled water
Dissolve sucrose in 15 ml of distilled water over
low heat.
1.Bring final volume to 25 ml with distilled water.
.Sucrose solution needs to be made fresh each
time you do this lab or frozen between uses to
prevent bacterial contamination.
.To check for contamination, swirl the bottles containing
sucrose or lysis buffer and look for floating particles and
bacterial strands.
(5)Proteinase K
(I)25 mg proteinase K
(II)2.5 mL distilled H2O
Add the water to the vial containing the
proteinase K powder.
Swirl to dissolve.
Freeze proteinase K solution between uses.
(6)1 M Tris (pH 8.0)
Tris (also known as THAM) is an abbreviation of
the organic compound known as tris
(hydroxymethyl) aminomethane, with the formula
(HOCH2)3CNH2 .
Tris is widely used as a component of buffer
solutions, such as inTAEandTBE buffer,
Preparation
especially for solutions ofnucleic acids.
12.1 g of Tris base is dissolved in 50 ml distilled
water and pH was adjusted to 8.0 with
concentrated HCl.
Final volume was made up to 100 ml with
distilled water and autoclaved.
(7)5M EDTA pH 8.0
EDTA is a widely used acronym for the chemical
compound ethylenediaminetetraacetic acid.
Its prominence as a chelating agent arises from
its ability to "sequester" di- and tricationic
metalions such as Ca2+ and Fe3+.
After being bound by EDTA, metal ions remain
in solution but exhibit diminished reactivity.
Chelation is thebindingor complexationof a bi-
or multidentateligand.
Chelants are chemicals that form soluble,
complex molecules with certain metal ions,
inactivating the ions
so that they cannot normally react with other
elements or ions to produce precipitates or scale.
The ion depletion is commonly used to
deactivate metal-dependent enzymes to
suppress damage to DNA (Protecting the DNA
from degradation).
Preparation
1.Add 18.6 g EDTA (disodium salt, m.w. 372.24) to
80 ml deionized or distilled water.
2. Adjust to pH by slowly adding approximately
2.2 g of sodium hydroxide pellets (m.w. 40.00).
If a pH meter is not available, adding 2.2 g of
NaOH pellets will make a solution of
approximately pH 8.0.
3. Mix vigorously with a magnetic stirrer or by
hand.
EDTA will only dissolve after pH has reached 8.0
(8)Tris-EDTA
(I)1 ml 1M Tris (TE) Buffer
(10 mM in solution) concentrated
HCl.
(II)200 l 0.5 M EDTA (1 mM in solution)
concentrated HCl.
(III)99 ml distilled water.
TE buffer is used by research laboratories to
store and protect DNA over long periods of time.
For our purposes, DNA can also be dissolved in
water and studied by agarose gel electrophoresis.
(9)5 M Sodium Chloride (NaCl)
(I)29.2 g NaCl.
(II)Distilled water.
Dissolve 29.2 g of NaCl in 80 mL of water over
low heat using a magnetic stirbar.
This is almost a saturated solution. It will take a
long time for the NaCl to dissolve completely.
Bring final volume to 100 ml.

(10) 0.9% Saline Solution or use


(I)9 g NaCl. Gatorade
(II)1l distilled H2O.
1.Mix in a clean container because will be putting
the saline solution in their mouths.
2.Swirl to dissolve.
Gatorade needs to be purchased fresh each time.
It will start to grow bacteria if stored open for
longer than a few days to a week.
Check for contamination by swirling the
container and looking for floating particles or
bacterial threads.
I prefer to use Gatorade because I know it's
sterile. Choose a light color flavor, such as lemon,
to keep from staining the DNA.
(11)7.5M NH4 acetate(MW = 77.08)

1.57.81g in 100ml ddH20


2.Autoclave and store at room temp.

(12)6M Guanidine hydrochloride


(MW = 95.53)

1.57.318 g in 100ml ddH2O.


2.Autoclave and store at room temp.
Equipments

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.