Bacterial Genomic DNA isolation practical ( Phenol-chloroform Method)
I have seen many people (including me) struggle to find a good practical protocol with principle, roles of reagents , observations and discussions all clubbed together as one. The literature is always dispersed throughout net across various papers and documentation. Therefore here I'm providing a summarized short but to the point practical for the Phenol-Chloroform Method of genomic DNA isolation from Bacteria(say E.coli).
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AIM
Isolation of genomic DNA
from bacterial cells
MATERIALS REQUIRED
LB medium (per ml, 10 mg tryptone, 5 mg yeast extract, and 5 or 10 mg
NaCl), Incubator
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Reagents |
20% SDS , Phenol: chloroform: isoamylalcohol
(25:24:1) , RNase , NaCl solution (5M) , chilled ethanol(70%) , TE(Tris-EDTA) buffer[10mM Tris-Cl(pH8.0), 1mM EDTA(pH 8.0] , Lysis buffer[TE buffer, proteinase K,
NaCl/SDS]
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|
Apparatus |
Eppendorf tubes, micropipettes , centrifuge
machine , vortex mixer , (Agarose)gel electrophoresis setup , UV transilluminator
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PRINCIPLE
Prokaryotic DNA is not
surrounded by a nuclear envelope , it is naked associated with some histone
like proteins in the cytoplasm of the bacteria called the nucleoid. For the
isolation of this DNA lysis of the cells must be done.
The isolation of bacterial
genomic DNA follows four broad steps:
·
Growing and Harvesting of Bacterial Culture- Here bacterial cell (say E.coli) culture is taken. The cells
were cultured in LB media at 37C, aerated by shaking at 150-250 rpm.
Appropriate cell density needed for use is checked by monitoring the Optical
Density (OD) at 600nm of the cell culture intermittently. Usually cells in mid
/ end log phase are chosen. Cells are harvested by transferring some of the
solution to a centrifuge tube or eppendorf tube and centrifuging at high speed for few minutes. The cells get
settled in pellet form which can further be resuspended for further use.
·
Bacterial cell lysis and Removal of Cell Debris- Cell lysis is carried out
by lysis buffer to rupture the cells in order to release their internal
contents comprising of proteins , nuclic acids and other molecules. A detergent
like SDS or CTAB is used to disrupt the cell membrane. Once
cell is disrupted, endogenous nucleases can cause extensive hydrolysis of the
DNA , which can be protected from endogenous nucleases by chelating Mg2++ ions
using EDTA.
·
Removal of contaminating biomolecules from the cell (RNA proteins lipids etc.)- Proteins associated
with DNA are denatured with SDS / Proteinase K and removed, this is done by the Phenol-chloroform-isoamyl
alcohol mix. The denatured proteins separate out and form a layer at the
interface between the aqueous and the organic phases which are removed after
centrifugation.
The
aqueous layer of chloroform contains our genomic DNA.
·
Precipitation , purification and visualization of DNA - Chilled ethanol is added
to cause DNA precipitation . The sample can be incubate under chill condition
if the precipitation is not instant or enough and then it is centrifuged.
The pellet is then washed and air dried. This dried sample is then resuspended in TE buffer and RNase is added ,incubated and then tracking dye is added for it to be visualized on electrophoresis gel and the DNA bands can be then visible as orange bands under UV illuminator.
ROLE OF REAGENTS
NaCl : Sodium
chloride which provides an osmotic shock to the cells causing them to burst
open. It also shields the negative charges of the phosphate backbone therefore
maintaining the integrity of the DNA structure.
Proteinase K :
is used for the destruction
of proteins in cell lysates and for the
release of nucleic acids . It effectively inactivates DNases and RNases.
Tris-EDTA
(TE) buffer : Tris helps to maintain a constant
physiological pH to prevent any damage to the DNA ; EDTA works as a chelating
agent , it chelates the metal ion(Mg2+) present as a cofactor in enzymes(like
DNases) and thereby inhibiting their action on the DNA.
SDS :
Sodium Dodecyl
Sulfate is an anionic detergent that
denatures secondary and non disulfide-linked tertiary protein structure,
altering their native shape. It also disrupts
the cell membrane and nuclear envelope , hence aiding in lysing cell during DNA isolation.
PCI : Phenol-chloroform-isoamyl
alcohol; It is a liquid-liquid extraction method in which the molecules
are separated based on their differential solubilities in the two different
immiscible liquids. In phenol-chloroform extraction of DNA, the solution is
added to aqueous solution of the lysed cells, the two phases are then
mixed, and the phases are separated by centrifugation. Centrifugation of this
solution mixture yields two phases: the lower organic phase and the upper
aqueous phase.
Since phenol is denser and non-polar, DNA is
immiscible in it and hence remains in the water/chloroform on top of phenol as
aqueous phase.
Chloroform increases the efficiency of phenol for
denaturing proteins and lipids and allows separation of the
organic and aqueous phase.
Isoamyl alcohol helps in reducing foaming between interphase.
RNase : It is added to hydrolyze RNA
molecules present in the solution so as to obtain a sharp band of DNA
only on the gel.
Ethanol : Solvents like ethanol have lower dielectric constant than water
i.e. they shield poorly against positive and negative charges in the solution.
Ethanol forms H bond with water molecules and shields the DNA from hydration .
This causes aggregation of DNA with positive ions in solution , precipitating
the DNA at the bottom.
Tracking dye
added to the sample prepared to track the sample on the Agarose gel and prevent
its overflow out of the gel. It contains Bromophenol blue(to provide color)
and glycerol(to provide density to the DNA so that it settles in the well ).
Bromophenol blue has slightly negative charge and hence moves in the same
direction as that of DNA.
EtBr
(Ethidium Bromide) is a DNA intercalating agent which is use to visualize the
DNA under UV. It is often added while preparing the agarose gel solution. Since
it is carcinogenic so should be handled with caution.
PROCEDURE
1. 1.5 ml of the overnight E.
coli(DH5a) culture is transferred to a 1.5 ml Eppendorf tube and
centrifuge at max speed for 1min to pellet the cells.
2. The supernatant was discarded and the pellet was resuspended in
600ul of lysis buffer and incubate at 37C for 1hr.
3. SDS was added and invert mixing was done till the solution become
viscous.
4. 600ul of PCI was added , invert mix and then centrifuge at high
speed for 5 min.
5. Two layers are visible with a white layer of proteins in the
middle. The upper aqueous layer is carefully transferred to a new eppendorf.
6. Step 4&5 can be repeated till the white layer is eliminated.
7. To precipitate the DNA 1 ml of 70% chilled ethanol was added and
the tube was mixed gently.[ Precipitation may be diffused so the tube can be
kept at -20C for min 30 min.]
8. The tube is then centrifuged at high speed for 15min at 4C.
9. Supernatant
was discarded and the DNA pellet was rinsed with
1 ml 70% ethanol (stored at RT) , spinned at max speed for 2 min,
supernatant removed and the DNA pellet
was left to air-dry .
10. Resuspended
the pellet in 50ul TE buffer (vortex if needed); 1-5 μl (1 mg/ml) RNAase was
added to it to completely remove RNA(incubate at 37C for 1hr). [Alternatively RNases can be added along with the lysis buffer]
11. Tracking dye was added and the sample was
subjected to gel electrophoresis.
STEPS SHOWING GENOMIC DNA ISOLATION FROM E.COLI CULTURE
OBSERVATION
The extracted
DNA was observed after running it on Agarose gel . A sharp band of genomic DNA
with characteristic orange glow in the transilluminator could be seen. A smear
of DNA is observed if shearing of DNA had occurred during isolation procedure.
Since RNase was added beforehand RNA band/ smear isn’t there.
DISCUSSION
Genomic DNA
that was isolated from E. coli cells
and run on gel was found close to the well forming a single sharp band because
of the large size of the DNA its mobility was hindered through the gel. There
can be poor yield of DNA owing to inconsistencies in drawing out the aqueous
layer while pipetting, DNA loss while decanting the supernatant , vigorous/harsh
mixing of Eppendorf after ethanol addition leading to shearing of DNA . Also if
enough incubation time is not given after addition of lysis buffer ; incomplete
lysis of cells would lead to lesser DNA yield and fainter bands on the gel.
Some methods
to ascertain the yield and purity of DNA extracted is:-
Absorbance
Method
DNA
absorbs light strongly at 260nm (A260), and the number generated allows one to
estimate the concentration of the solution. However, DNA is not the only molecule that absorbs UV light
at 260nm. RNA also absorb at 260nm, and the aromatic amino acids like tyrosine
and tryptophan present in protein absorb at 280nm. If these contaminants are
present in the DNA solution, will contribute to the total measurement at 260nm.
To evaluate DNA purity, the ratio of the absorbance at 260nm divided by the
reading at 280nm is done. Good-quality DNA will have an A260/A280 ratio of
1.7–2.0. Reading lower than 1.7 means that there are much contaminants present
in the solution. Ratio 1.8 is ideal for DNA.
A260/230 ratio is also used as a secondary measure to
check nucleic acid purity. It is used to indicate the presence of unwanted
organic contaminants such as Trizol, phenol, Guanidine HCL and guanidine
thiocyanate.
Florescence
Method
Fluorescence
methods are more sensitive than absorbance, especially for low-concentration
samples, and the usage of DNA-binding dyes allows precise measurement of DNA concentration.
Fluorescence measurements
are set at excitation and emission values that vary depending on the dye
chosen. The concentration of unknown samples is calculated by comparison to a
standard curve generated from samples of known DNA concentration.
Gel
Electrophoresis Method
Agarose
gel electrophoresis can be used to check the yield and concentration of DNA by
comparing the intensity of the DNA sample on the gel with known DNA standards.
The DNA size can be known by comparing to a DNA ladder run simultaneously with
the sample.
PRECAUTIONS
o
All
reagents should be freshly prepared.
o
Ethidium
bromide should be handled with care.
o Pipetting and aqueous layer separation should be done carefully.
References
https://microbenotes.com/protocol-phenol-chloroform-extraction-of-prokaryotic-dna/
https://vlab.amrita.edu/?sub=3&brch=77&sim=218&cnt=1
https://www.elabprotocols.com/protocols/#!protocol=184
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