INTRODUCTION

BLOOD
a bodily fluid in animals that delivers necessary
substances such as nutrients and oxygen to
the cells and transports metabolic
waste products away from those same cells
composed of blood cells suspended 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 or sonicating the
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 of nucleic
acid
rapidly inactivates nucleases that 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
phenol denatures proteins in the sample
denaturated proteins stay in organic phase
aqueous phase containing nucleic acid is mixed
with the chloroform

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 of agarose
biomolecules separated by applying an electric
field to move the charged molecules through
an agarose matrix
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

Transfer
Supernatant into a
new tube
-Add 100l Chloroform/ Isoamly alcohol/ Phenol
(25:24:1)
- Shake by hand for 10 minutes
- Centrifuge at 3000 rpm for 5 mins at 10 C
Transfer Again the
supernatant into a
new tube
-Add 100l chloroform/ Isoamylalcohol (24:1)
-Shake by hand for 10 minutes
-Centrifuge at 3000 rpm for 5 mins
at 10 C

Transfer the
Supernatant in to
a new tube
-add 30 l 3 M sodium acetate (pH 5.2) and 100 l
isopropanol
-Shake gently until the DNA precipitated
Use a glass
pipette and
make a hook
over the Bunsen
burner and
capture the DNA

Wash the DNA in

70% Ethanol

Dissolve the DNA in 50100 l TE- buffer

overnight at 4C on a
rotationg shaker

Measure the DNA

concentration in a
Spectrophotometer and
run 200 ng on a 1 %
Agarose gel

RESULTS AND
DISCUSSION

GUIDE
QUESTIONS

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?