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4. PLANT GENOMIC DNA ISOLATION
The application of molecular biology techniques to the analysis of complex
genomes depends on the ability to prepare pure, high molecular wt. DNA.
Various methods for the isolation of genomic DNA are available. The
selection of any particular protocol depends on the type of tissue used.
The present protocol describes the isolation of plant genomic DNA by CTAB
method. CTAB is a cationic detergent that has the property of precipitating
nucleic acids and acidic polysaccharides from solutions of low ionic
strength. Under these conditions proteins and acidic polysaccharides
remains in solution. In solutions of high ionic strength, the CTAB forms
complexes with proteins and all but the most acidic polysaccharides; but
will not precipitate nucleic acids. CTAB is therefore particularly useful
for purification of DNA’s
from organisms that produce large quantities of polysaccharides eg; plants.
Materials:
I 5M NaCl Stock 100 ml
29 gms of NaCl is dissolved in 100 ml of dis. H2O and
sterilized by autoclaving.
II.
0.5M EDTA – 8.0 Stock 100 ml
18.61 gm of EDTA is dissolved in 100 ml of dis. H2O. Adjusted to
pH 8.0 with NaoH and sterilized by autoclaving.
III. 1M Tris 7.57 Stock 100 ml
12.11 gm of Tris is dissolved in 100 ml of dis. H2O. Adjusted to
pH 7.5 with conc. HCl and sterilized by autoclaving.
IV.
CTAB extraction buffer
Stock 50ml 100ml 200ml 300ml 400ml
450ml 500ml 600ml
dH2O
36.5 73 146 219 292 330
365 440
1M Tris 5.0 10.0 20.0 30.0
40 45.0 50.0 60.0
5M NaCl 7.0 14.0 28.0 42.0
56 63.0 70.0 84.0
0.5M EDTA 1.0 2.0 4.0 6.0
8.0 9.0 10.0 12.0
14 MBME2 0.5 1.0 2.0
3.0 4.0 4.5 5.0 6.0
CTAB3 0.5g 1.0g 2.0g 3.0g
4.0 4.5g 5.0g 6.0g
1 – Use freshly made
2 – Add BME to warmed buffer 960-65°C),
just prior to use. (ß mercaptoethanol). Usually obtained as a 14.4M
solution.
3.
CTAB – Cetyl trimethyl ammonium bromide.
V.
Tris satured phenol
Before use, phenol must be equilibrated to a pH >7.8 because DNA will
partition into the organic phase at acid pH. Liquified phenol should be
stored at –20°
C. Before equilibration phenol is warmed to room temperature and them melted
to 68° C. To the melted phenol 0.1% of
hydroxyquinoline is added Hydroxyquinoline is an antioxidant. A partial
inhibitor of RNase and a weak chelator of metal ions. In addition its
yellow colour provides a convenient way to identify the organic phase.
An equal volume of 0.5M Tris cl pH 8.0 is added and the mixture is stirred
on a magnetic stirrer for 15’
minutes. When the two phases have separated the upper (aqueous) phase is
aspirated using a glass pipette attached to vacuum line quipped with traps.
To the phenol equal volume of 0.1M Tris cl (pH 8.0) added; stirred on a
stirrer, upper phase is aspirated. The above step is repeated until the pH
of the phenolic phase is >7.8. After the phenol is equilibrated
and the final aqueous phase has been removed, 0.1 volume of 0.1M Tris cl (pH
8.0) containing 0.2% β-mercaptoethanol is added.
The equilibrated phenol is stored in this form under 100 mM Tris cl
(pH 8.0) in a light-tight bottle at 4° C for
periods of upto one month.
VI.
Chloroform: Isoamyl alcohol:
A mixture consisting of equilibrated phenol and chloroform: isoamyl alcohol
(24:1) is prepared and stored under 100 mM Tris cl (pH 8.0) in a light-tight
bottle at 4°
C for periods of upto one month.(Neither chloroform nor isoamyl alcohol
requires treatment before use).
VII. RNase
RNase is dissolved in 10 mM Tris cl (pH 7.5) and 15 mM NaCl at a
concentration of 10 mg/ml, heated to 100°C
for 15; cooled slowly to room temperature, dispensed into aliquots and
stored a –20° C.
VIII. 100% Ethanol:
Distilled ethanol
IX. 70% Ethanol:
contains 70 ml of 100% ethanol and 30 ml of dis H2O
Protocol:
·
9ml of warm (65°
C) CTAB extraction buffer is added to 300-400 mg ground lyophilized tissue
in a 15 ml polypropylene centrifuge tube. The tissue is distributed along
the sides of the tube before adding the buffer to avoid clumps of dry tissue
in the bottom and mixed by gentle inversion.
·
The mixture is incubated for 60-90 min at 65°
C with continuous gentle rocking.
·
Tubes are removed from the oven and 4.5 ml of Chl/isoamyl alcohol (24:1) is
added and rocked gently for 5-10 min.
·
Centrifuged at 1500g for 10 min at room temperature
·
Top aqueous layer is pipetted into a new 15 ml tube containing 50 ul of 10
mg/ml RNase A, mixed by gentle inversion and incubated for 30’
at RT.
·
To the above equal volumes of phenol:chl isoamyl alcohol is added; mixed
gently and centrifuged at 1500 g for 10 min at room temperature.
·
Top aqueous layer is transferred into a new tube and extracted once again
with phenol; Chl:
isoamyl alcohol
·
Centrifuged at 1500g/10’/RT
·
The top aqueous layer is transferred to a new tube and 0.6 volumes of
isopropanol is added and mixed by gentle inversion.
·
The precipitated DNA is hooked out with a glass rod
·
Washed with 70% ethanol, dried at 65°
C and dissolved in 100 ul of T10E0.1.
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