The document describes the phenol-chloroform method for extracting DNA from bacterial cells. This method uses a series of steps: 1) bacterial cells are centrifuged and washed, 2) SDS and proteinase K are added to lyse cells and degrade proteins, 3) NaCl and CTAB are added to precipitate DNA, 4) chloroform-isoamyl alcohol is added to remove lipids and proteins, 5) isopropanol is added to precipitate DNA, which is then washed with ethanol and resuspended in TE buffer. The phenol-chloroform method separates DNA from other cellular components based on differential solubility in aqueous and organic phases.

1. By
PHENOL-CHOLOROFORM
METHOD

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.