This document discusses methods for extracting nucleic acids like DNA and RNA from samples. It describes the key steps in nucleic acid extraction as isolation, purification, and extraction to separate nucleic acids from other cellular components. The main steps are cell lysis to break open the cell, precipitation to separate nucleic acids from debris, and purification to further clean the nucleic acids. Both physical and chemical methods are used, with choices depending on sample type, nucleic acid type, and intended downstream use. Common extraction techniques discussed include phenol-chloroform, salting out, and using proteinase K or magnetic beads.

1. Nucleic acid
Isolation
Nucleic
acid
Isolation

2. DNA isolation :
*
aims to collect as much DNA
DNA purification :
*
Reduce contamination from isolated DNA
DNA extraction :
*
achieve both isolation and purification

3. Is a method to purify DNA by using physical and /or
chemical methods from a sample Separating DNA
from cell membrane , proteins, and other cellular
components.
What is extraction

4. Molecular studies , diagnosis , forensic science , vaccine
development , pharmaceuticals.
Sample source : microorganism , human , plant
sample type : Blood ,urine ,biopsy ,sputum, hair ,bones
,semen
Nucleic acid type ; RNA , DNA
Planned usage : PCR , sequencing ,fingerprinting
Importance and application of DNA , RNA extraction
choosing best extraction method depend on ;

5. DNA.
Found within cell
Eukaryotic : inside nucleus
Prokaryotic : inside cytoplasm
Inside cells with enzymes ,protein
,organic and in organic compound
all-non nucleic acid component
need to be removed

6. Steps in DNA extraction :
1.lysis
2.precipitation
3.purification

7. Physical method
Mechanical disruption
plant cells, tough cell wall
blender
Mortar And pestle
Cutting
sonicator
1.Cell lysis/cell disruption
Chemical method
Detergents :
Sodium Dodecyl Sulfate (SDS)/lauryl
Sulfate (rapid disruption
Enzymes:
Target aminoacids bonds
ProteinaseK;animal cell
Cellulase;plant cell
lyticase: yeast
Lysozyme:gram positive bacteria

8. 2. Precipitation
Separate DNA from cellular debris
after lysis : cell extract :DNA mixed with cell
debris and contaminants ( proteins,
detergents,reagents,etc)
Sodium (Na+)ions. Neutralize negative
charge
More stable
Alcohol: isopropanol/ethanol
cause precipitation
DNA insoluble with alcohol
Protease : degrade DNA associated proteins
Filtration

9. 3. Purification
After precipitation ;
DNA separated from
aqueous phase
Isopropanol or
absolute ethanol
Removal of cellular
debris and unwanted
material
Storage and handling

10. Extraction methods :
chemical DNA extraction
method
Physical DNA extraction
method
Organic DNA extraction
method
Inorganic DNA
extraction method
Magnetic bead. DNA
Extraction
Paper DNA extraction
method
Phenol-chloroform
DNA
Extraction method
Salting out method
Silica gel based DNA
extraction method
Proteinase k DNA
extraction method

11. Organic
DNA
extraction
Plant sample

12. 1.Lysis : Extraction buffer
Detergent : disruption
of cell membrane
Reducing agent :protein
denaturation
Chelating agent
:removal of Mg++ions
Buffer
Salt
Sodium dodecyl sulfate(SDS )
Cetyl Trimethyl ammonium bromide(CTAB)
B mercaptoethanol
EDTA
Tris : pH8
Nacl

13. Step 1
Cell
Lysis
Disruption of cell wall
Esp for plant
E.g: blender , mortar and pestle
,liquid nitrogen
Protein digestion
Transfer ground plant samples
into eppendorf
tube
Extraction buffer
Lyse cell membrane and inhibit
nuclease activity

14. .incubate at 60deg celsius for 20-
40 mins in a water bath
centrifugation
10000rpm,10mins
removal of cell debris
transfer supernatant (crude lysate )
to a new tube

15. 2.Precipitation
Crude lysate: with nucleic acids and other cell constituents
add equal
amount of
phenol -
chloroform
to the
crude
lysate
*Mix by
vortex
centrifugati
on
10.000rpm
10mins
separates
aqueous
phase ,
interphase
and organic
phase
collect
aqueous
phase (with
nucleic acids
) into new
tube
Do not
disturb the
organic
phase
Use wide
bore tips
:avoid
mechanical
DNA
damage
add ice cold
95%ethanol
and salt to
aqueous
phase
Mix by
inversion
-20 deg
celsius
1hour -
centrifugati
on
10.000rpm.
10min

16. 3.Purification
Decant supernatant
*
wash DNA pellet with 70% ethanol
*
mix by inversion
*
centrifugation 10.000 rpm5mins
*
Discard ethanol (noresidual ethanol)
*
*Dry pellet at RT for 30 mins
*Dissolve and resuspend DNA pellet : TE buffer /
nuclease free water
DNA pellet

18. In organic DNA
extraction
(proteinase k
method)
Blood sample

19. Lysis
,Add 2ml blood
with 10-20UL TE
buffer
centrifugation at
2500rpm for
20mins
add 10-15UL TE
buffer to pellet incubate at high
temperature
lyse cell and
nuclear
membrane
Digest protein
discard
supernatant
Mix gently 60-65 deg celsius
2hours

20. centrifugation
2500rpm for
15mins
Add proteinase k
solution and DNA
extraction buffer
incubate at high
temperature
Proteinase k
discard
supernatant.
60-65 deg celsius
2hours
60 deg c(37-70deg )
PH 8.0

21. Precipitation
Add 1-2 ml chilled isopropanol and a pinch of Nacl
mix by invertion
Centrifugation at 8000-10000rpm
discard supernatant

22. purification
Add 1ml ethanol to pellet
centrifugation 10000 rpm for 1-2mins
Add TE buffer and incubate at water bath for 2-3 hours
Discard ethanol
Dry pellet