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INTRODUCTION
BLOOD
abodily fluidinanimalsthat delivers
necessary substances such asnutrientsand
oxygento thecellsand transportsmetabolic
wasteproducts away from those same cells
composed ofblood cellssuspended in blood
plasma
Whole blood plasma and cells together

DNA EXTRACTION
process of isolating of DNA from sample using
a combination of physical and chemical
methods.
Follows the process of
Cell lysis
DNA purification
DNA resuspension
AGE

CELL LYSIS
to expose the DNA within the cell
removal of membrane lipids
commonly achieved by chemical and physical
methods-blending, grinding orsonicatingthe
sample
Cell lysis buffer
Detergents
Surfactants

PROTEIN PRECIPITATION
Degradation of proteins associated with DNA
Achieved by the addition of a protease
Protease K - digest protein and remove
contamination from preparations ofnucleic
acid
rapidly inactivatesnucleasesthat might
otherwise degrade the DNA or RNA during
purification
aided by the addition of a salt such as
ammonium or sodium acetate

PHENOL-CHLOROFORM EXTRACTION
phenoldenaturesproteins in the sample
denaturated proteins stay in organic phase
aqueous phase containingnucleic acidis
mixed with thechloroform

DNA RESUSPENSION
DNA is insoluble in the alcohol and will come
out of solution
alcohol serves as a wash to remove the salt
previously added
DNA can be re-suspended in a buffer such as
Tris or TE
TE - solubilize DNA or RNA, while protecting
it from degradation

AGAROSE GEL ELECTROPHORESIS


separate a mixed population of DNA or
proteins in a matrix ofagarose
biomolecules separated by applying
anelectric fieldto move the charged
molecules through anagarosematrix
biomolecules are separated by size in the
agarose gel matrix

MATERIALS &
METHODOLOGY

MATERIALS

Whole Blood

Phenol / chloroform / isoamyl alcohol (25:24:1)

Chloroform / isoamyl-alcohol (24:1)

Sodium acetate, (CH3COONa) 3M

Sodium dodecyl sulfate, (SDS) 20%

Proteinase K (10mg/ml)

Isopropanol (2-Propanol)

Lysis buffer

SE buffer

TE buffer

MATERIALS

Phenol

Ethanol, 70 %

Agarose gel, 1%

Ice

EQUIPMENT
Dryblock heater
Refrigerated centrifuge
Uv-vis spectrophotometer
Microcentrifuge tubes
Micropipettors and tips

100ul blood + 300ul


lysis buffer shake
30 mins. on ice
centrifuge at 1200
rpm, 10 min., 4 degC.

Remove supernatant
+ 100ul lysis buffer
resuspend pellet
centrifuge centrifuge
at 1200 rpm, 10 min.,
4 degC.

Remove supernatant

Remove supernatant
+ 50ul SE-buffer
resuspend pellet
centrifuge centrifuge
at 1200 rpm, 10 min.,
4 degC.

Add 50ul SE-buffer


resuspend the pellet
add 0.5ul proteinase K
(10mg/ml) + 2.5 ul 20%
SDS shake incubate
overnight at 37 degC. In a
water bath

Add 100ul phenol


shake
by hand for 10 min.
centrifuge at 3000 rpm,
5min., 10 degC.

AGAIN: Supernatant new


tube + 100ul Phenol /
chloroform / isoamyl
alcohol (25:24:1)
shake
by hand for 10 min.
centrifuge at 3000 rpm,
5min., 10 degC.

Supernatant new tube


+ 100ul Phenol /
chloroform / isoamyl
alcohol (25:24:1) shake
by hand for 10 min.
centrifuge at 3000 rpm,
5min., 10 degC.

Transfer supernatant
new tube + 30ul 3M
sodium acetate ph 5.2 +
100ul isopropanol shake
gently DNA precipitated
capture DNA: hook glass
pipette

Wash DNA in 70% ethanol


dissolve in 50-100ul TEbuffer overnight, 4 degC.
In a rotating shaker

Store the rest of the DNA


at -20 degC.

Measure DNA in
spectrophotometer
200
ng on 1% agarose gel

RESULTS AND
DISCUSSION

GUIDE
QUESTION
S

1.) WHAT IS THE ROLE OF THE


FOLLOWING IN DNA EXTRACTION?
A. Ethanol wash to remove salt previously added
B. NaCl Degradation of proteins associated with
DNA
C. SDS Cell lysis; detergent
D. TE Buffer - solubilize DNA or RNA, while
protecting it from degradation
E. EDTA CELL LYSIS: chelates cations that may
bind to the negatively-charged DNA

2.) GIVE THE COMPLETE CHEMICAL


NAMES OF THE FOLLOWING:
A. SDS Sodium dodecyl sulfate
B. Tris Buffer tris (hydroxymethyl)aminomethane
C. EDTA Ethylenediaminetetraacetic acid

3.) WHY SHOULD THE EXTRACTED DNA


BE IMMERSED IN TE BUFFER?
The purpose of TE Buffer is to solubilize DNA while
protecting it from degredation.

4.) WHAT IS THE ABSORBANCE OF


DSDNA AT 260NM? SSDNA?