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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
MATERIALS &
METHODOLOGY
MATERIALS
Whole Blood
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
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.
Transfer supernatant
new tube + 30ul 3M
sodium acetate ph 5.2 +
100ul isopropanol shake
gently DNA precipitated
capture DNA: hook glass
pipette
Measure DNA in
spectrophotometer
200
ng on 1% agarose gel
RESULTS AND
DISCUSSION
GUIDE
QUESTION
S