2. A phenol-chloroform extraction is a liquid-
liquid extraction. A liquid-liquid extraction is a
method that separates mixtures of molecules
based on the differential solubilities of the
individual molecules in two different immiscible
liquids. Liquid-liquid extractions are
widely used to isolate RNA, DNA, or proteins
3. DNA extraction is done for:
1.Tissue typing for organ transplant.
2.Detection of pathogens
3.Human identity testing
4.Genetic research
5.PCR
6.Restriction fragment length
polymerization.
7.Hyberdization method.
4. Cell cycle has four phases
1. Lag phase
2. log phase
3. Stationary phase
4. Death phase
5. Primary metabolites are produced
during log phase and secondary
metabolites are produced during
stationary phase.
Cells for DNA extraction are
extracted from log phase and early
stationary phase.
6. Step1:
For DNA extraction 1.5ml culture is taken in
appendroff tubes. It is centrifuged at 10,000
rpm for 3 minutes until pellets are formed.
Step 2:
Supernatant is discarded
Step3:
Cell pellets are washed with 300 µl 8.1 TE
buffer.
Mix well by vortex.
7. TE buffer is composed of
1. TRISMA
2. EDTA
TRISMA : maintains the pH of TE buffer at 8.1
EDTA: chelates Mg++ ions due to which cell
membrane structure is distorted.
8. Step4:
After washing with TE buffer, 30 µl
SDS solution and 3 µl proteinase- k is added.
After adding both of them mix them
thoroughly by doing vortex. Incubate it at 37ºC
for 1hour In water bath.
A. Proteinase-K:
Proteinase-K is used to degrade
proteins.
9. B. SDS: Sodium dodecyl sulfate.
1. Is anionic solution.
2. It causes cell lysis. Attack membrane
lipids.
3. It also denatures proteins and lipids.
10. Step5:
Now add 100µl 5M Nacl and 80µl CTAB.
Mix thoroughly and incubate tubes at 65ººC
for 10 minutes.
A. Nacl: mask phosphate to bring DNA
close together to form aggregates.
11. B. CTAB: CETILE TRIMETHYL AMONIUM
BENZOATE
1. Binds with DNA and form insoluble
bonds.
2. Denatures lipid and proteins of cell.
12. Step 6:
Add 700-800µl chloroform isoamyl alcohol and
centrifuge it for 15 minutes.
Chloroform: Binds with lipids and protein to
precipitate them.
Isoamyl: Anti foaming agent.
13. After centrifugation two phases are formed.
1.Upper layer:
Upper layer is called aqueous layer
that contains DNA, RNA and Plasmid.
2. Lower layer:
Is organic layer which contains proteins
and lipids
14. Step 7:
After centrifugation aqueous phase is
transferred to another appendroff tube.
To this tube add 700-800 µl phenol
chloroform isoamyl alcohol. Centrifuge
again for 15 minutes.
15. PHENOL:
Precipitates protein and lipids
CHLOROFORM:
Chloroform binds to non aqueous
compounds i.e. lipids and proteins
and precipitate them.
ISOAMYL:
Its an anti-foaming agent.
16. Step 8:
After centrifugation again transfer aqueous
layer into another eppendorf tube. Add
600 µl isopropanol to it and again centrifuge
for 15 minutes.
ISOPROPANOL:
Dehydrates DNA and forms weak DNA
pellets.
17. Step 9:
After centrifugation remove supernatant
and wash pallets with 70% ethanol.
ETHANOL:
Pellets produced by ethanol are stronger
but less number of pellets are produced.
18. Step 10:
Supernatant is removed and pallets are
air dried.
Add 100 µl TE buffer . Aqueous phase
contains isolated DNA.
Use isolated strains for subsequent
experiment or store at 20ºC.
19. Always add Proteinase-K before
adding SDS because SDS causes
bacterial culture to become viscous.
After addition of SDS solution
becomes clear.
Many DNA isolation methods don’t
require vortexing because of
shearing of DNA but in vortexing for
2 to 3 minutes can cause protein
denaturing.
