ISOLASI DNA

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DNA and RNA EXTRACTIONS
DNA and RNA EXTRACTIONS
A protocol / method / schedule /procedure for extraction / isolation of both DNA and
RNA from the same material typically plant leaf / leaves
(See also DNA Isolation protocol)
1) Take one medium sized leaf or half a large leaf (5 to 20 cm^2), weigh and freeze
in liquid nitrogen.
2) 2) Grind the tissue in a bleached and baked pestle and mortar with liquid
nitrogen.
3) 3) Transfer the powder produced to a l5ml Falcon blue cap tube. Add 2 ml of
homogenization buffer (4 ml per g) and disperse the tissue in it.
4) 4) Leave for 10 min at room temperature.
5) 5) Add 2 ml of phenol/chloroform and vortex.
OR,
1) Put a small or medium sized leaf into a 4" x 6" 500 guage (thick!) plastic
bag.
2) Add 2-3 ml homogenization buffer (you may need more for large leaves) to
the bag.
3) Grind the leaf inside the bag using the top end of a 50 ml corex tube.
4) Pour the homogenate immediately into a 15 ml Falcon blue cap tube
containing equal volume of phenol/chloroform then vortex.
5) Goto step 6
6) Spin for 10 min at 2,500 rpm in a bench centrifuge.
7) Transfer 0.7 ml of the aqueous phase to an Eppendorf tube, add 0.7 ml
phenol/chloroform, vortex and spin for 5 min.
8) Transfer 0.6 ml of the aqueous phase to a fresh Eppendorf tube, add 0.6 ml
phenol/chloroform, vortex and spin for 5 min.
9) Transfer 0.5 ml of the aqueous phase to a fresh Eppendorf tube, add 0.5 ml
chloroform/isoamyl alcohol, vortex and spin for 5 min.
10) Add 4M LiCl to the final conc. of 2M and leave for several hrs at 4oC (better
o/n). Spin for 10-15 min. RNA should be in a pellet. Remove supernatant
and precipitate the DNA with ethanol.
11) Treat RNA fraction with DNase RNase-free, and the DNA fraction with
RNase-free of DNase.
Notes:
Homogenization buffer:
0.1 M NaCl 2% SDS
50mM Tris-HCl pH 9.0 10 mM EDTA
(ideally DEPC treated water and everything else RNase free)
then add 0.1 mg/ml proteinase K { added fresh }

Pat Heslop-Harrison University of Leicester December 2003.

(I'm afraid I did not note the source of this protocol. Alex Vershinin first used
for oil palm DNA and RNA extraction to look at retroelement activity - see
Kubis SE, Castilho AMMF, Vershinin AV, Heslop-Harrison JS. 2003.
Retroelements, transposons and methylation status in the genome of oil palm
(Elaeis guineensis ) and the relationship to somaclonal variation. Plant
Molecular Biology 52 : 69-79. doi:10.1023/A:1023942309092)

DNA isolation & extraction

CTAB TECHNIQUE / Method / Schedule / Protocol FOR DNA ISOLATION /
DNA EXTRACTION FROM PLANT LEAF / LEAVES SAMPLES
(see also DNA RNA double isolation procedure if both DNA and RNA are needed)
Reagents needed
CTAB buffer
2% CTAB 20gm CTAB
20mM EDTA 40ml EDTA stock (0.5M)
100mM Tris-Cl pH 8.0 100ml Tris-Cl stock (1M)
1.4M NaCl 280ml NaCl stock (5M)
make up to 1 Litre with water, pH 7.5 - 8.0, and autoclave
+ 0.2% Mercaptoethanol
Wash Buffer
76% Ethanol
10mM NH 4 Ac
DNA Extraction
1. Preheat 5ml CTAB (add 10l mercaptoethanol to each 5ml CTAB) in a blue-topped
50ml centrifuge tube at 60-65 o C. Remove and discard midribs, and wrap laminae
in aluminium foil and freeze in liquid nitrogen. 0.5 1.0 gm tissue/5ml CTAB (Can
store leaf material after liquid Nitrogen 1-2 days at 20 or 80 for longer periods)
2. Gently crumble leaf tissue over cold pestle of liquid nitrogen. Grind frozen leaf with
one spatula of fine sand add 0.5 spatula of PVPP powder after grinding.
3. Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid
leaving dry material around rim of tube. Adjust CTAB volume to give a slurry-like
consistency, mix occasionally.
4. Incubate for 60 min at 60 o C
5. Add equal volume of chloroform/iso-amyl alcohol (24:1), Mix for about 3min, then
transfer contents to narrow bore centrifuge tubes. Balance by adding extra chlor/iso.
Spin 5,000rpm for 10min (ensure correct tubes used), brake off. (For extra pure
DNA isolation - spin and retain supernatant before chloroform extraction).
6. Remove supernatant with wide-bore pastette (cut off blue tip) to clean tube, repeat
chloroform extraction once. Supernatant should be clear, though may be coloured.
7. Precipitate DNA with 0.66 vol. of cold isopropanol - can leave overnight. Spool out
or spin down DNA, 2min at 2,000rpm.
8. Transfer to 5ml wash buffer for 20min.
9. Dry briefly and resuspend in 1ml T.E. (can be left overnight)

10. Add 1l 10mg/ml RNAse to each 1ml T.E./DNA mixture and incubate for 60min at
37 o C. (If RNase in the sample doesn't matter stages 11 and 12 may be omitted)
11. Dilute with 2 volumes TE and add 0.3vol 3M Sodium acetate (pH 8) + 2.5 vol cold
100% ethanol,
12. Spool DNA out. Air dry and resuspend in 0.5 to 1ml TE or water (takes time) and
freeze until required.
DNA Quantification
An approximate way to determine DNA concentration is to look at the viscosity of the
solution: not accurate to 10% but, unlike spectrophotometry, you will not get results
which are 10 or 100 times wrong!
In a microcentrifuge tube, DNA solutions stronger than 0.1 ug/ul will show a reluctance
to pour when you tilt the tube. From about 0.5 ul/ug and above, you can tilt the tube very gently - and the solution will stay at the end. If you dip a 10-200 ul (yellow) pipette
tip into the solution and pull it away, a solution of 1ug/ul will form a distinct string from
the surface to the tip which breaks when about 1 to 2 mm long.
Make a 0.8% agarose gel with 1x TAE and 0.1l of Ethidium bromide (10mg/ml) per
10ml solution. Load samples undiluted and at a 1 in 10 (1+9) dilution., with 3l loading
buffer. Also include a Lamda ladder cut with HindIII and EcoRI. This contains 100ng of
DNA per microlitre and use as follows:
1l ladder + 4l water + 2l loading buffer
2l ladder + 3l water + 2l loading buffer
The different bands of the ladder are of known molecular weight and known DNA
concentration. Match the brightness of your samples with those of the two dilutions of
the ladder. Refer to the diagram to match the band with the concentration. Remember
that although the ladder concentrations are absolute, you have loaded 5l of sample and
also diluted some of them. This must be taken into account when calculating the
strength of the sample s in ng/l.
Pestles and mortars washed for 20-30min in 0.25M HCl, rinsed in water and air-dried,
all mess to be tidied up and tubes washed and left to drain.
Pat Heslop-Harrison, Trude Schwarzacher, John Bailey University of Leicester
2003/2009. See www.methods.molcyt.com

A PLANT NUCLEAR DNA PREPARATION

PROCEDURE
PMB Newsletter (1981) Vol. II
J. Bedbrook, CSIRO Division of Plant Industry, PO Box 1600, Canberra City ACT
2601, AUSTRALIA
This method can be used to prepare Petunia cv. Rose du Ciel nuclear DNA suitable for
restriction enzyme analysis and cloning. Chloroplast DNA contamination will vary
depending on the genotype. Greater chloroplast DNA contamination results when this
method is used with the "Mitchell" line of Petunia.
1. Young leaves (20 g) from 1-3 month old Petunia plants are ground at 4 C with a
minimal volume of 0.3 M sucrose, 5 mM MgCl2, 50 mM Tris HCl (pH 8) (HB) in a
mortar and pestle. The slurry volume is brought to 100 ml with HB and filtered
through 2 layers, then 4 layers of cheesecloth.
2. The filtrate is centrifuged at 1000 g for 5 min and the pellet resuspended in 100 ml
of HB and repelleted at 1000 g for 5 min. This pellet is resuspended in 100 ml of
HB containing 2% Triton X-100 incubated at 4 C for 10 min and centrifuged at 1000
g for 5 min. The pellet is washed with 100 ml of HB with Triton X100,
recentrifuged, and the pellet cleaned of excess liquid with a paper towel.
3. The pellet is resuspended in 16 ml of 30 mM Tris-HCl (pH 8), 10 mM EDTA (RB),
and transferred to a 100 ml flask. 2 ml of 10% (w/v) Sarkosyl is added along with 2
ml of 5 mg/ml pronase. The mixture is heated at 60 C for 5 min and incubated with
gentle shaking at 37 C for 5-10 hrs. The solution is highly viscous and contains
among other things, starch grains and nuclear debris.
4. After the incubation, measure the total volume of the lysate and bring it to a volume
of 20 ml, Add 20 g of CsCl and dissolve completely over a period of about two
hours at 4 C.
5. Bring the mixture to room temperature, add 2 ml ethidium bromide (10 mg/ml). mix
in gently and thoroughly.
6. Dispense in centrifuge tubes and centrifuge at 35K rpm for 30 hrs at 15 C. Collect
the DNA band under long wave uv light.
7. Recentrifuge the DNA and collect, extract the ethidium bromide with RB-saturated
isoamyl alcohol and dialyze against 5 mM Tris-HCl (pH 8), 0.25 mM EDTA. Store
at 4 C.

PLANT DNA ISOLATION

1. Prepare 5-20 g of clean, frozen, young leaves taken from plants grown under
controlled conditions and exposed to darkness for two days prior to isolation.
Remove mid-ribs.
2. Grind leaves in stainless steel blender containing 150-200 ml of ice cold H buffer at
maximum speed for 1 min.
3. Pour the homogenate in a 250 ml centrifuge bottle (on ice) while filtering through
one layer of miracloth (Calbiochem) under four layers of cheesecloth (all previously
wetted with 10 ml clod H buffer).
4. Centrifuge at 2000 g, 4 C, 20 min.
5. Discard the green supernatant and resuspend the pellet in 40 ml ice cold HT buffer.
6. Transfer to a 50 ml teflon tube (Oakridge OK) and centrifuge at 2000 g, 4 C, 10
min. Repeat until pellet of nuclei becomes greyish-white (1-3X). If anthocyanins are
present in the plant, the pellet will be reddish-brown.
7. Resuspend the pellet thoroughly in 12 ml of HT buffer then add 12 ml of lysis
buffer.
8. Immediately, add 23.28 g of powdered CsCl and incubate the tubes at 55-60 C for
one hour with occasional inversion.
9. After solubilization of the CsCl, centrifuge the tubes at 28000 g, 15 C, 30 min.
10. Filter the supernatant through two layers of cheesecloth into a 38 ml quick-seal tube
containing 1.47 ml EtBr solution using a 50 ml syringe and a 16 G needle as a
funnel. Complete volume with CsCl solution.
11. DNA is recovered after centrifugation using standard procedures.
1X TE: 10 mM Tris-HCl (pH 8), 1 mM EDTA
1X H: 10X H, 400 ml; sucrose, 684 g; -mercaptoethanol, 8 ml; water to 4000 ml [pH
9.5]
Lysis buffer: Na-sarcosine, 4 g; Tris base, 2.42 g; Na2EDTA, 2.98 g; water to 200 ml
[pH 9.5]
10X H: spermidine, 20.35 g; spermine, 27.8 g; Na4EDTA 83.24 g; Tris base, 24.2 g;
KCl, 119.2 g; water to 1900 ml [pH 9.5]; Add Phenyl methyl sulfonyl fluoride (PMSF)
solution [7 g in 100 ml of 95% ethanol]
1X HT: 1X H, 1000 ml; Triton X100, 5 ml
CsCl solution: CsCl, 97 g; 1X TE, 100 ml
EtBr solution: EtBr, 1 g; water, 100 ml