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2 Isolation of Plant DNA (Murray and Thompson 1980)

2 Isolation of Plant DNA (Murray and Thompson 1980)

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Isolation of Plant DNA


2. Chloroform and isoamyl alcohol solution in the ratio of 24:1

3. 3 M Sodium acetate (pH 5.2):

Sodium acetate ¼ 408.1 g + Sterile H2O ¼ 800 ml

Adjust pH to 5.2 with glacial acetic acid and make up the volume to 1 l and

autoclave the solution before use.

4. DNase-free RNase A:

RNase A (10 mg/ml) in 10 mM Tris (pH 7.5) + 15 mM NaCl

Heat to 100 C for 15–20 min to make it DNase free and cool slowly to room

temperature (RT). Store in small aliquots at À20 C.

5. Proteinase K: 20 mg/ml proteinase K (store at À20 C)

6. 25% SDS

SDS – 25 g

Sterile H2O – 100 ml (warm to dissolve)

7. TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0

Tris – 1.211 g

EDTA – 0.372 g

Sterile H2O – ~800 ml

Adjust pH to 8.0 and make up the volume to 1 l with sterile H2O


1. Material for DNA extraction

Procure the seed material which will be the source of DNA. Wash the seeds

thoroughly with distilled water and then with 50% ethanol for 10 min. Soak the

seeds in 0.001% mercuric chloride for 10 min and then wash them several times

with distilled water and soak them overnight. Keep the seeds for germination on

wet germination towels at the desired temperature and humidity till they grow

3–6 in. in height. Cut the seedling 1 in. above the surface to minimize the

bacterial contamination and cut them into smaller pieces. Weigh them and

store them after a dip in liquid N2 at À20 or À70 C for further use.

2. Isolation of plant DNA

1. Add b-mercaptoethanol (b-ME) to the required amount of 2Â CTAB

extraction buffer to a final concentration of 0.2%. Heat b-ME/CTAB solution

to 65 C in a waterbath for 5 min.

2. Grind 10 g of etiolated seedlings in liquid N2 to a fine powder with a pestle

and mortar. Be very careful while powdering the tissue as the mortar and

pestle can shatter due to the extreme cold.



Techniques in Molecular Biology

3. Transfer the ground tissue (frozen powdered form) to a 500-ml conical flask

containing the preheated b-ME/CTAB extraction solution (50 ml)

and incubate the mixture for 45–60 min at 65 C with occasional mixing

(Tissue:Extraction buffer : 1:5).

4. Extract the homogenate with equal volume of chloroform solution by gentle

mixing (10 min).

5. Centrifuge for 5 min at 12,000 rpm at 4 C.

6. Transfer the upper (aqueous) phase to a fresh tube with a wide bore pipette

tip and re-extract with chloroform solution.

7. Centrifuge as above and transfer the aqueous phase to a fresh tube.

8. Precipitate the DNA with 0.6–1 vol. of isopropanol (Mix well) at À20 C for

atleast 30 min (precipitating overnight substantially increases the yield).

9. Centrifuge at ~12,000 rpm for 15 min at 4 C.

10. Wash the pellet (at least twice – necessary to remove traces of CTAB and

chloroform) with 70% ethanol and dry it until all visible traces of ethanol are

gone. Re-suspend the pellet in minimal volume of TE buffer (0.1–0.5 ml per

gram of the starting material). Do not overdry the pellet.

11. For removal of RNA, to the re-suspended DNA add 20 mg/ml DNAse-free

RNAse and incubate in a waterbath at 37 C for 1 h.

12. For removal of proteins, to the above mixture add 100 mg/ml of proteinase

K solution, 1/10 volume 3 M sodium acetate (pH 5.2), and 1/100 volume

of 25% SDS. Mix well and incubate at 60 C for 1 h.

13. Extract once with saturated phenol, then twice with 24:1, chloroform:

isoamyl alcohol.

14. Precipitate the DNA using 2 volumes of 95% chilled ethanol. Wash the

pellet twice with 70% ethanol and dry it partially. Re-dissolve it in TE buffer

and check the purity of DNA.

3. Purity test of DNA

(i) Spectrophotometric test

In a spectrophotometer, check the optical density (OD) of a dilution of the

DNA preparation at 260 and 280 nm. Pure DNA has an A260/A280 ratio of

1.8–2.0 in 10 mM Tris-Cl, pH 8.5. Strong absorbance at 280 nm, resulting in

a low A260/A280 ratio, indicates the presence of contaminants such as


We can use conversion factor 50 to convert O.D. to concentration in

mg/ml as DNA at a conc. of 50 mg/ml has an absorbance of 1 at 260 nm.

(ii) Agarose gel electrophoresis

Electrophoresis of DNA samples on 0.8% agarose gel with DNA markers

and their staining with ethidium bromide and examination under UV should

reveal a large band migrating close to the origin.


Isolation of RNA


Problems During Extraction (DNA isolation and storage)






DNA degradation due to old tissue; senescence (in vivo)

Improper storage of tissues (slow freezing, freeze-thaw)

Breakage during isolation

Breakage after isolation (freeze-thaw, nuclease or bacterial contamination,

particularly in dialysis)






Use fresh tissue.

Store properly.

Extract gently.

Be certain that the dialysis tubing has been treated appropriately, store DNA

clean and frozen. Test storage solution (TE) for nuclease activity. When the

sample is degraded, use frequent-cutting enzymes to reduce the average size of

fragments compared, thereby minimizing degradation effects. If the sample has

been contaminated, clean by (1) velocity gradient centrifugation, (2) Phenolchloroform extraction, or (3) commercially available wash solutions.


Isolation of RNA (Brawerman 1974)

Ribonucleic acid (RNA) occurs as ribonucleoprotein particles in intact cells.

The total RNA includes three classes – ribosomal, messenger, and transfer RNA.

There are a number of methods described for the extraction of total RNA and the

method selected largely depends upon the source material and the experimental

purpose for which RNA is extracted. Among the available methods used with plant

materials, the phenol-chloroform method is the common method used to recover the

total RNA intact.


The ribonucleoprotein complex is dissociated by SDS into RNA and protein,

deproteinized by phenol and the free RNA left in aqueous solution is precipitated

in the cold after adding alcohol.


Magnetic stirrer

Bench top centrifuge

Cold room (4 C)

Phenol (freshly redistilled)

Extraction buffer (pH 9.0)



Techniques in Molecular Biology

Tris-HCl (0.1 M) – 1.21 g

NaCl (0.075 M) – 0.44 g

EDTA Na2 (0.005 M) – 0.19 g

Water to – 100 ml

• Ethanol

• SDS 10% (w/v in water)

• Ether


All operations need to be conduced at 0–4 C.

1. Freeze 0.5–5 g of the material in a mortar and pestle with liquid N2, grind to a

fine powder, then to a paste and extract using 10 volumes of extraction buffer.

2. Centrifuge the homogenate at 2,000 Â g for 3 in.

3. Transfer the supernatant to a volumetric flask and stir with 0.1 volume of 10%

SDS for 2–3 min.

4. Add an equal volume of buffered phenol (freshly redistilled phenol saturated

overnight with 100 mM Tris-HCl pH 8.5).

5. Partition the content by centrifuging at 2,000 Â g for 5 min and collect the

upper aqueous phase into a separate flask.

6. Shake the lower and interphase again with an equal volume of extraction buffer

for 5 min and centrifuge.

7. Combine the aqueous phase with the first one (step 5) and stir with an equal

volume of buffered-phenol for 5 min.

8. Repeat the extraction and centrifugation steps at least 5 times or until the

interphase shows no proteins.

9. Finally, collect the upper aqueous phase containing RNA, dissolve in it about

250 mg NaCl, add two volumes of cold ethanol (96%), and leave the flask

overnight at À20 C for RNA precipitation.

10. Collect RNA by centrifugation at 2,000 Â g for 10 min. Wash the pellet (RNA)

with 70% ethanol, ethanol, ethanol:ether (1:1 v/v), and finally with either.

Dry the pellet gently in vacuo for a few minutes.

11. Dissolve the RNA completely in elution buffer for further analysis by


12. Dilute 20 ml aliquot to 2 ml with buffer and read the absorbance using 1 cm

light path cuvette at 260 nm in a spectrophotometer. One A260 unit is assumed

equivalent to 40 mg RNA/ml. Otherwise, the RNA content is estimated colorimetrically (see estimation of RNA).


• All the glassware and solutions should be sterile. Any contaminating RNase is

inactivated by rinsing the glassware with 1% diethyl pyrocarbonate solution.

• Phenol: Chloroform (1:1) mixture is an effective deproteinizing agent that

retains the poly (A) tail in mRNA intact.

• Any contaminating DNA will appear just like cotton wool during ethanol addition.


Quantitative Estimation of DNA


• Use freshly distilled colourless phenol. It can be stored frozen in small aliquots

in brown-coloured bottles. Wear gloves while handling phenol.

• A variety of extraction medium, chelating agents, deproteinizing agents, etc. are

used for the extraction of RNA.

• The denatured proteins gather at the interphase after low-speed centrifugation

which should be discarded.

• The volume of aqueous phase will be drastically reduced if the phenol is not

fully saturated.

• Deproteinization using phenol leads to the loss of poly (A)-tail of mRNA



Quantitative Estimation of DNA (Burton 1956)

The quantitative estimation of DNA could be carried out by number of methods;

one of the method is described below.


Under extreme acid conditions, DNA is initially depurinated quantitatively followed

by the dehydration of sugar to o-hydroxylevulinylaldehyde. This aldehyde

condenses, in acidic medium, with diphenylamine to produce a deep-blue coloured

condensation products with absorption maximum at 595 nm.


• DNA standard (0.5 mg/ml).

• Saline citrate solution (0.15 M NaCl, 0.015 M Na3 citrate).

• Diphenylamine reagent: Mix 5 g fresh or recrystallized diphenylamine, 500 ml

glacial acetic acid, and 13.75 ml conc. H2SO4. Stable for 6 months at 2 C; warm

to room temperature and swirl to remix before use.


1. Prepare separate marked tubes containing 1, 2, and 3 ml aliquots of the isolated

DNA dissolved in standard saline citrate and similar aliquots of a 0.5-mg

DNA/ml standard.

2. Make all sample tubes, and a separate blank, up to 3 ml with H2O.

3. Add 6 ml of diphenylamine reagent to each tube, and after mixing, heat the tubes

in a boiling water bath for 10 min. Cool the tubes.

4. Read the absorbance of blue solution at 600 nm against the blank.

5. Construct a standard graph A600 (ordinate) vs. quantity of DNA (abscissa) and

then calculate the concentration of DNA dissolved in the saline citrate solution.

This method is commonly applied for samples of 50–500 mg DNA.




Techniques in Molecular Biology

Quantitative Estimation of RNA (Ashwell 1957)

Methods suitable for pentose determination are used for measurement of RNA,

which include reactions with orcinol, phloroglucinol, aniline, etc.


The method depends on conversion of ribose (pentose) in the presence of hot acid

to furfural, which then reacts with orcinol to yield a green colour. The colour

formed largely depends on the concentration of HCl, ferric chloride, orcinol, the

time of heating at 100 C, etc. up to certain maxima.


• Standard RNA.

• Sample RNA solutions.

• Orcinol acid reagent: Add 2 ml of a 10% solution (w/v) of FeCl3.6 H2O to

400 ml of conc. HCl.

• 6% Orcinol: Dissolve 6 g orcinol in 100 ml 95% ethanol. It is stable for 1 month.

Refrigerate in a brown bottle until use.

• Colorimeter.


1. Prepare a standard RNA (50 mg RNA/ml) solution in ice-chilled 10 mM

Tris-acetate, 1 mM EDTA buffer (pH 7.2), or any other suitable buffer by

dissolving RNA completely.

2. Dissolve the isolate RNA in the above buffer solution to an approximate

concentration of 50 mg/ml.

3. Prepare a series of tubes containing 0.5, 1.0, 1.5, and 3.0 ml of isolated RNA,

0.5, 1.0, 1.5, and 3.0 ml of 50 mg standard RNA/ml.

4. Make up each tube to 3.0 ml with water. In addition, set a blank containing

3.0 ml of water.

5. Add 6 ml of orcinol acid reagent to each tube.

6. Add 0.4 ml of 6.0% alcoholic orcinol to each tube. Shake the tubes to mix the

contents, and then heat all tubes in a boiling water bath for 20 min.

7. Cool the tubes, and read the absorbance at 660 nm against the blank.

8. Draw a standard curve using A660 and the concentration of standard RNA.

Calculate the amount in the isolated RNA solution using the graph.

• The yield and purity of RNA preparation can be assessed by measuring the

absorbance of ultraviolet light by a solution of nucleic acid. A pure RNA

solution should give a 260 nm:280 nm of 2; 1 U of A260 measured in 1 cm

light path length is equivalent to 40 mg/ml.


Southern Blot Analysis of Plant DNA



Southern Blot Analysis of Plant DNA

This procedure is used for hybridizing labelled-DNA probes to DNA fragments

separated on an agarose gel and then blotted on nitrocellulose (or nylon) membrane.

Southern blot analysis involves the following steps.

1. Restriction (digestion) of DNA

Plant DNA is restricted using restriction endonucleases. The restriction endonucleases used are type II enzymes which recognize either 4, 5, 6, or 8 bp long

sequences and generate either 30 protruding ends, 50 protruding ends, or bluntended fragments. The next step is the separation of restricted fragments using

agarose gel electrophoresis.

2. Agarose gel electrophoresis

Agarose gel electrophoresis is used to separate DNA molecules according to

their size. The negatively charged DNA molecules migrate in an electrical field

from negatively charged cathode to positively charged anode, with smaller

molecules migrating faster than the bigger ones through the pores of the matrix.

The pores of the matrix can be changed by altering the agarose concentration.

After electrophoresis, DNA fragments are visualized by staining with ethidium

bromide which fluoresces in UV.

3. DNA transfer to nylon membranes

DNA hybridization requires the breakage of hydrogen bonds between the

complementary strands of DNA, which can be achieved either by high temperature treatment or treatment with denaturing agents like formamide. These

treatments cannot be done on agarose gels. Hence, DNA is transferred from

the gel onto a synthetic membrane. Nylon coated with nitrocellulose is used

which combines the physical strength of nylon membrane with high resolution

of nitrocellulose membrane. The banding pattern is preserved during the transfer. Since only single-stranded DNA binds to nitrocellulose, the DNA has to be

denatured before transfer. The weak interaction between the DNA and the

membrane achieved during transfer is further modified into a covalent body by

heating to 80 C.

Solutions and Reagents

(a) Restriction of DNA

1. Mix in a sterile 1.5 ml Eppendorf tube

DNA – 5 mg

10Â buffer – 5.0 ml

Restriction enzyme – 2.5 ml (10 units/ml)

Sterile water to – 50 ml

2. Incubate the mixture at 37 C for 5 h overnight.

3. Add 5 ml of 10Â dye to the sample, centrifuge for 5 s in an Eppendorf

centrifuge tube, and load on an agarose gel.



Techniques in Molecular Biology

(b) Agarose gel electrophoresis

1. 10Â TBE buffer

Tris base – 108 g

Boric acid – 55 g

0.5 M EDTA (pH 8.0) – 20 ml

Dissolve in DDW and make up to 1 l. Autoclave before use.

2. Gel-loading buffer (10Â dye)

Glycerol – 5 m

10Â TBE – 1 ml

Bromophenol blue saturated – 1 ml

Xylene cyanol (10%) – 1 ml

Add DDW and make up to – 10 ml

Mix well. Divide into 1 ml aliquots, autoclave, and store at –20 C.

(c) DNA transfer to nylon membranes

1. 0.25 M HCl

Conc. HCl – 21.5 ml

H2O – 978.5 ml

2. Denaturing solution (1 l)

(1.5 M NaCl and 0.5 M NaOH)

NaCl – 87.66 g

NaOH – 40.0 g

Add DDW – to 1 l

3. Neutralizing solution

1 M Tris-Cl (pH 8.0) + 1.5 M NaCl

Tris base – 121.12 g

NaCl – 87.66 g

Adjust pH to 8.0 with HCl and make up the volume to 1 l with DDW

and autoclave before use.

4. 20Â SSC

(3 M NaCl + 0.3 M Sodium citrate, pH 7.0)

NaCl – 175.32 g

Na citrate – 88.213 g

DDW – to 1 l

Adjust pH to 7.0 with citric acid and make up the volume to 1 l with DDW

and autoclave before use.


Southern Blot Analysis of Plant DNA



A. Agarose Gel Electrophoresis (0.8%)

1. Weigh 0.8 g agarose and put in a 250-ml conical flask. Add 100 ml of

1Â TBE buffer and gently boil the solution in a microwave oven with

occasional mixing until all agarose particles are completely dissolved.

Allow it to cool to 50 C. Add ~10 ml of ethidium bromide (10 mg/ml) and

mix well. Prepare the gel mould and keep the comb in position. Pour the

cooled gel solution into the gel mould and allow the gel to set for 20 min.

2. Fill the horizontal electrophoresis chamber with 1Â TBE. Remove the comb

from the gel and place gel with the tray in the electrophoresis chamber.

3. Load the digested DNA sample carefully into the wells. In one well, load a

standard marker (Lambda DNA restricted with HindIII)

4. Run the gel at 20 mA overnight.

5. Stain the gel in 100 ml sterile distilled water containing 1 mg/ml ethidium

bromide for 10 min. Briefly destain with sterile water (ethidium bromide may

be added directly to the gel solution prior to pouring or to the running buffer).

6. Visualize the DNA bands on a UV transilluminator and place a ruler next to

the gel to be able to determine the fragment sizes later on. Take a picture for


Problems and Solutions During Digestion Process





Endonuclease or exonuclease contamination

Titrate enzymes properly; switch to cleaner enzymes.

Partial digestion

Use more enzyme; two-step digestion.

Electrophoretic Artefacts











Retardation due to excess DNA

Use less DNA; use purified or semipurified organellar DNA

Unclear bands

Reduced buffering capacity of running buffer (make new buffer)

Missing small bands

Reduce electrophoresis time or use a combination of agarose and polyacrylamide gels.

Missing large bands

Too much BSA in digests; could also be due to DNA degradation.

Non-specific background (i.e. flecking) in gels loaded with end-labelled

samples (particularly agarose gels)

Use higher grade agarose, making certain that it is completely dissolved;

make sure plates and apparatus are clean; rinse gels before drying down.



Techniques in Molecular Biology

B. Transfer of DNA to Nylon Membrane (Reed and Mann 1985; Ausubel et al.


1. After staining and photography of the gel, dip and shake it in 200 ml of

0.25 M HCl for 10–15 min at room temperature in a glass baking dish until

the bromophenol blue barely turns yellow (Acid depurinates the DNA,

breaking large fragments into smaller pieces for more efficient transfer).

2. Decant the HCl and rinse with distilled H2O for 1 min and denature the

DNA by soaking the gel in several volumes of 1.5 M NaCl and 0.5 M NaOH

for 1 h at room temperature with constant shaking.

3. Decant the denaturing solution, rinse the gel with distilled H2O for 1 min

and neutralize the gel by soaking in several volumes of 1 M Tris-Cl (pH 8.0)

and 1.5 M NaCl for 1 h at room temperature with constant shaking.

4. Wrap a piece of Whatman 3MM paper around a piece of glass plate and

place it inside a large baking dish. Fill the dish with 20Â SSC almost to the

top (20Â SSC is 3 M NaCl + 0.3 M sodium citrate, pH 7.0) and smooth all

air bubbles in the 3 MM paper with a glass rod.

5. Invert the gel so that its original underside is now upper most and place it

on the wet 3 MM paper. Remove air bubbles, if any, between the gel and

the paper.

6. Cut a piece of nylon membrane about 1–2 mm larger than the gel. Dip it in

sterile water (>20 min) and then float the membrane in 20Â SSC for

5–10 min.

7. Place the wet nylon membrane on top of the gel and remove the air bubbles

that are trapped between the gel and the membrane (Air bubbles trapped

between the gel and membrane cause uneven or incomplete transfer).

8. Wet two pieces of Whatman 3 MM paper, cut to exactly the same size as the

gel in 20Â SSC, and place it on top of the nylon membrane followed by

6 cm stack of dry paper towels. Put a glass plate on top of the stack and

weigh it down with 500 g weight.

To prevent short circuiting of fluid between the paper towels and 3 MM

paper under the gel, surround the gel with a watertight border of Saran wrap

(Do not touch nylon membrane with naked hands. Always wear gloves and

use forceps while handling nylon membranes. Ethidium bromide is


9. Allow transfer of DNA to proceed for 6 h to overnight. Small fragments of

DNA (>1 kb) takes 15 h or more.

10. Disassemble the blot in reverse order, and using a soft pencil, clearly label

the slots on the membrane. Also mark the filter to define its orientation

relative to the gel.

11. Soak the membrane in 10Â SSC at room temperature for 5–10 min.

12. Place the filter on 3 MM paper to air-dry it.

13. Bake the membrane at 80 C for 1 h in a vacuum oven.

14. Soak the membrane in warm 2Â SSC, seal it in plastic bag, and store in the

dark in refrigerator until use.


Southern Blot Analysis of Plant DNA


C. Preparation of Probe by Oligolabelling (Using Hexalabel DNA Labelling Kit)

This method relies on the priming of the polymerase reaction on the template

DNA with random hexanucleotide primers. The complementary strand is

synthesized from the 30 end of the primer with the help of the large fragment

of DNA polymerase I, Exonuclease Minus (Klenow Fragment, exo-) in the

presence of labelled deoxyribonucleoside triphosphates.


1. Add the following components into 1.5 ml microcentrifuge tube

DNA template (100 ng) – 10 ml

Hexanucleotide in 5Â reaction buffer – 10 ml

Deionized water up to – 40 ml

Vortex the tube and spin down in a microcentrifuge for 3–5 s. Incubate the tube

in a boiling water bath for 5–10 min and cool it on ice. Spin down quickly.

2. Based on labelled triphosphate (dATP or dCTP), use Mix A or Mix C,


3. Add the following components in the same tube:

Mix A (or Mix C) – 3 ml

[a-32P]-dATP [a-32P]-dCTP**

(1.85 M Bq ¼ 5 mCi)

Klenow fragment, exo- (5 U) – 1 ml

Shake the tube and spin down in a microcentrifuge for 3–5 s. Incubate for 10 min

at 37 C.

4. Add 4 ml of dNTP and incubate for 5 min at 37 C.

5. Stop the reaction by the addition of 1 ml 0.5 M EDTA, pH 8.0

6. The labelled DNA is used directly for hybridization or stored at À20 C.

Removal of the unincorporated label is not necessary for most applications,

since the levels of its utilization are usually high. If required, the unincorporated

dNTP can be removed by chromatography on SephadexTM G-50 or by selective

precipitation of DNA with ethanol in the presence of ammonium acetate.

Removal of Unincorporated Label (Optional)

1. Add 1 volume of 4 M ammonium (pH 4.5) to the labelled DNA, mix and vortex.

2. Add 2 volumes of ethanol, mix and chill in ice for 15 min.

3. Heat at 37 C for 2 min to re-dissolve free deoxyribonucleotides precipitated with

occasional mixing.

4. Centrifuge at 12,000 Â g for 15 min and carefully aspirate the supernatant.

5. Wash the pellet once in 90% ethanol and dry the pellet.

6. Re-dissolve the labelled DNA in TE buffer and use the probe for hybridisation.

Determination of Per Cent Incorporation (DE 81 Filter Binding Assay)

1. Dilute 1 ml of the labelling reaction with 0.2 M EDTA (1:100). Spot 3 ml

(in duplicate) of the diluted sample on Whatman DE 81 circular (2.3 cm) filters.

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