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GENOMIC DNA EXTRACTION FROM RICE LEAVES
(MODIFIED CTAB METHOD-mini-prep)
1, Cut 1-2 leaves in 2ml tube and put 2 sterilized steel balls. Arranged the
prepared tubes in a genogrinder rack and place the rack in bucket and pour
liquid Ninto it (its better if the tubes were immersed in Liquid N- 30seconds).
Place it in genogrinder about 2-3 minutes or until totally grind.
Grinding tissues in liquid nitrogen disrupts the cells.
2. Add 800ul of 2X CTAB buffer warmed to 65°C. Mix thoroughly. Incubate at
65°C for 30 minutes to 1hour (with mixing from time to time).
One of the most common problems in DNA extraction in some species of
plants is the presence of high concentrations of polysaccharides which co
purify with DNA in normal phenol-chloroform extractions. DNA isolation from
these species is preferably achieved by CTAB (cetyltrimethyl ammonium
bromide) treatment. The DNA is soluble in the presence of CTÁB at high salt
concentration (0.7 M). A CTAB-nucleic acid precipitate will form if the salt
concentration drops below 0.4 Mat room temperature. Ata salt concentration
of 0.7 M, CTAB forms complexes with cell wall debris, other polysaccharides
and proteins. This protein/polysaccharide complex is removed by chloroform
extractions leaving DNA in the aqueous phase to be precipitated with ethanol.
CTAB- a cationic detergent, destroy the membranes, denature and
disassociate proteins from DNA
Sarkosyl and SDS- anionic detergent
Raising the ionic strength by the addition of 5M NaCl provides an environment in
which DNA-protein interaction, which are often brought about by ionic
interactions are thermodynamically neutralized by the presence of other ions.
This helps to dissociate nuclear protein (especially histone) from DNA
Amajor consideration in any DNA 0solation procedure is the inhibition or
inactivation of DNases which can hydrolyze DNA. The buffer in which the cells
are suspended should have a high pH (8.0 or greater) which is above the
optimum of most DNases. Suitable buffer system based on Tris-HCL pH 8 -9 -
maintain the pH range to avoid the activity optima of degrading enzymes.
EDTA 0S also included in the resuspension buffer to chelate divalent cations
(such as Mg++) which are required by DNases. DNase activity is further
controlled by keeping the tissues and reagents cold and a heating step that will
thermally denature DNase (but must not be hot enough to denature the DNA)
3. Cool briefly and add 800ul chloroform-isoamyl alcohol (24:1). Shake at room
temperature for 20 minutes. Spin at 12,000 rpm for 10 minutes.
Proteins are extracted with chloroform-isoamyl alcohol. Chloroform-isoamyl
alcohol is used to deproteinize (it is called the "Sevag procedure" after the
1
chloroform causes surface
person whofirst developed and used it in 1938). The
denaturation of proteins; the isoamyl alcohol reduces foaming, aids separation
solution.
and maintains the stability of the layer of the centrifuged, deproteinized
The solution also removes lipids and some polysaccharides.
4. Decant aqueous phase (top phase) into a new 2 mlL tube.
for 30
5 Add ice-cold 400 mL isopropanol (1 volume) and incubate at -20°C
minutes to 1 hour (or overnight).
The nucleic acids (including RNA) are precipitated with isopropanol.
6 Spin at 12000 rpm for 10 minutes and decant isopropanol and wash pellet
with 70% ethanol and drain dry. Dissolve pellet in TE (200 uL). Add 2 uL of
RNAse (10 mg/mL) and incubate at 37°C for 30 minutes.
RNAse is added to degrade contaminating RNA. Optimum activity of RNAse
is achieved at 37°C.
7 Add 1/10 volume of 3M sodium acetate (20 uL). Add 2 volumes of ice-cold
absolute ethanol (400ul). Incubate at -20 Cfor 1hour or overnight.
In this precipitation, the ribonucleotides from RNase treatment will remain in
solution loaving purified DNA in the pellet. The pellet can then be dissolved in an
appropriate buffer.
Ethanol lowers the effective water concentration. causing large biomolecules
to interpenetrate and aggregate. The resu is a visible precipitate at the
interface, where the ethanol is concentrated. As DNA is precipitated and
removed, more is exposed to the ethanol and will precipitate.
Sodium acetate adjusts salt concentration (final concentration of sodium
acetate is 0.3 M).
8 Spin tubes at 12,00 rpm for 5 minutes. Drain and rinse pellet with 70%
ethanol. Air dry. Dissolve pellet in 100-200 uL TE (depends on the size of the
pellet). Irncsbate at 65°C to facilitate suspension of DNA pellet.
Washing with 70% ethanol reduces residual salt.
9. Dilute as necessary and store the stock solution in -20°C and the working
solution at 4°c.
Storing DNAin TE inhibits the activity of DNAses.
Young leaves- meristematic leaves are small, more nuclei per gram of tissue
Old leaves- more starches,phenolic build-up,tissue become tougher, difficult to lyse the
cell
2
QUANTITATION OF DNA
method of
of DNA, the simplest
1. pure solutions nm where an OD of 1 in a 1 cm path
Spectrophotometric. Forabsorbance
reading the at 260 single-stranded DNA
quantitication double-stranded DNA., 40 ug/mnl for
length = 50 ug/ml for oligonucleotides. An absorbance ratio of 260 nm
ug/ml for and RNA
and RNA and 20-33an estimate of the purity of the solution. Pure DNA
and 280 nm gives method S hot
ODeo/O Dogn Values of 1.8 and 2.0, respectively. This
Solutions have
useful for small quantities of DNA or RNA (<1 pg/ml).
DNA + 995 uL ddHa0. Check absorbance at 260nm
Dilute DNA sample: 5 uL
and 280 nm.
Use ddH,0 as blank
Nanodrop (latest spectrophotometric)
nanodrop and the machine will read the
Just put 2ul of stock DNA in the
concentration of the sample(s)
also be checked through
2. Agarose gel electrophoresis. The quality of the DNA can
agarose gel electrophoresis.
For DNA solution of unknown concentration, mix 2 uL of DNA with 3 uL
loading dye. Load onto 1% agarose gel (3 grams agarose/300 ml of buffer 1X
TBE (Tris borate EDTA), Include as control different concentrations of DNA
lambda (25, 50, and 100 ng). Run the gel at 100V for 45 minutes to 1 hour.
Stain in SyberSafe solution for 20-30 minutes and view the gel under the gel
documentation system. Compare intensity of the samples with the standards
(known concentrations of DNA lambda) to estimate concentration.
Loading Dye
The 10x Gel Loading Buffer is intended to be mixed with samples containing DNA in
order to facilitate loading samples into the wells of horizontal and vertical agarose and
polyacrylamide gels. The addition of Gel Loading Buffer increases the density (glycerol)
of the sample and gives it color, thus facilitating loading. In addition, it contains two dyes
that migrate in the same direction as the nucleic acids, thus serving as rough indicators
of the electrophoretic progress. The 10x concentrate is composed of 0.21%
Bromophenol Blue, 0.21% Xylene Cyanol FF, 0.2 MEDTA, pH 8.0 and 50% Glycerol.
Solutions.
"X" 3. Percentagesolutions.
PercentagePercent 2. CX. PREPARATION
SOLUTIONS OF
several Many
Concentrations. B.concentrationtimesMany nolar A.
liter Calculation
to
Preparation TOmake
enzyme
the its
V=Cx buffers
V=Cf=concentrated Ex. Ex. =
molecular
29.29 solution
final finalinitial V= buffer,concentration
To bring To(v/v)(wlv)
up
in of
buffers make
Prepare of set the up g100 of
vol, To = in
concentration, vol, Cfx molecularWorking
avoid the up volume = weight. isMolar,
or orV, stock buffer volume a weight 100 ml one
other a are 0.7%
buffers volume amountwhere
having of ml of in %
restriction prepared inthe (ml) (g) a
solutionsbiology to
solution sol 5M whichand"X"
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with of or of C; working 100 in mole
to reaction1X. is NaCl
solution
desired concstock = make ml 100 100
fresh initial and require components, digestion as liter liter 1
Solutions.
with of mlml solution
of from concentrated
solution) agarose of
solution
double-distilled concentration,
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solution
solution. solution
the
Concentrated =
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needed
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ul, solutions, contains
from water are
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would of the
a diluted e.g. weight NaCI) number
of final
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water. of
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prepare
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4
SOLUTIONS NEEDED
2X CTAB (1Liter) facilitate
water. Heat and stir to
CTAB- dissolve in 860 mL sterile distilled
20 g
dissolution.
Add:
81.82 g NaCl
100 mL 1M Tris pH 8.0
40 mL 0.5 M EDTA, pH 8.1
Autoclave and store at room temperature
just before use (optional).
40 uL B-mercaptoethanol per 20 mL solution
1Liter of TE Buffer (10 mM Tris, 1mM EDTA)
10ml of 1 MTris solution (pH 8.0)
2 ml of 0.5 M EDTA (pH 8.0)
988 ml sterile water.
Or
(disodium) to 1 liter of distilled water. Adjust pH
Add 1.21 g Trizma base and EDTA
using HCI and autoclave the solution.
1M Tris-HCI, pH 7.6, 8.0, 8.5, 9.0, 9.5:
121.1 g Tris base
ddH20 to 800 ml
ddH20 to 1 L.
Adjust pH with concentrated HCl and then add
tetraacetate):
0.5 M EDTA, pH 8.0 (disodium ethylenediamine
186.1 g Na2EDTA
NaOH. EDTA will not go
Dissolve in approx. 400 ml ddH2O, adjust pH to 8.0 with 10N
with distilled water and
into solution until the pH is about 8.0. Adjust to 1 liter final volume
autoclave.
5
3M NaOAc, pH 5.2
408.24 g NaOAc-3H20
with glacial acetic acid and bring to
sove in apprOx. 800 ml ddH20, adiust pH to 5.2
a final volume of 1 Lwith ddH20. Sterilize by autoclaving.
RNase A
DISSOlve pancreatic RNase at a concentration of 10 mg/mL in 10 mM Tris-HCI, pH 7.0
uL 1M Tris-CI, pH 7.5 + 15 uL of 1 M
and 15 mM NaCI (975 uL sterile ddH20 + 10Puncture the lid with a needle to allow
NaCi). Place in sterile microcentrifuge tube.with sufficient water on a hot plate and heat
Steam to escape. Place the tube in a beaker minutes. Remove beaker from the hot plate
until boiling (100°C). Continue heating for 15 the water bath. Allow both to cool slowy to
and leave the tube containing the RNase in store at -20°C.
room temperature. Aliquot into 1.5 mL microfuge tubes and
EtBr stock
10 mg/ml in dH20. Store in a light-tight container (A bottle or a
tube wrapped aluminum
foil)
SyberSafe stain
50ml distilled water/nanopure water + 5ul SyberSafe
5M NaCIl (sodium chloride)
292.20 g NaCl
ddH20 to 1000 ml
10% Ammonium persulfate (APS)
5g APS
ddH20 to 50 ml (store at 4°c).
Agarose Gel Electrophoresis Buffers for Analysis of DNA
Buffer Composition of working solution Components
Stock solution components per liter
TAE lx 40 mM Tris-acetate 50x Tris base 242 g
Glacial acetic acid 57.1 ml
1mM EDTA 100 ml
0.5 M EDIA, pH 8.0
TBE 5x Tris base 54 g
0.5x 45 mM Tris-borate
Boric acid 27.5 g
1mM EDTA
20 ml
8MNG2EDTA
Components per 10 ml
Gel loading 0.25% bromophenol blue Bromophenol blue 25 mg
buffer 0.25% xyene cyanol FF Xylene cyanol FF 25 mg
40% [w/v) sucrose* Sucrose*
* 15% Ficoll (Type 40O) or 30% glycerol can be used instead of sucrose.
Reagents 500 mL 1Liter 2Liters
50 X TAE
Trizma base 121.0g 242.0 g 484.0 g
Glacial acetic acid 28.55 g 57.1 g 114.2 g
0.5 M EDTA 50 ml 100 ml 200 ml
Autoclave
10 X TAE
Trizma Base 54.0 g 105.0 g 216.0 g
Boric Acid 27.5 g 55.0 g 110.0 g
EDTA 4.65 g 9.3 g 18.6 g
Or 0.5 M EDTA 20 ml 40 ml 80 ml
10 XTBE Buffer
Trizma Base 60.2g 121 g 242 g
Boric acid 30.5g 61g 122 g
EDTA 4.5g 9g 189
