Experiment 8

Objective: To isolate genomic DNA from plant leaves.

Principle: The efficiency of isolation of genomic DNA depends upon:

• The lysis of cells to release cells contents.
• Removal of contaminants such as RNA and proteins and concentration of
DNA.
In order to prepare a cell extract, Tris-Cl buffer is the actual buffering component.
Ethylene diamine tetrachloroacetic acid (disodium salt) (Na2EDTA) chelates Mg++
ions that are essential for overall structure of cell envelope and for deoxyribonuclease
(DNase) activity. Sodium dodecyl sulfate (SDS) aids the process of lysis by removing
lipid molecules and thereby causing disruption of cell membrane; it also denatures
proteins; β-mercaptoethanol (β-ME or 2-ME) acts as reducing agent and reduces
disulfides and thus disrupt the tertiary structure of proteins and denatures them.
Solution is then neutralized with potassium acetate (KoAC) solution. This solution
precipitates denatured proteins along with SDS. Sample is centrifuged to remove this
insoluble cell debris and the resulting supernatant containing nucleic acid.
Isopropanol precipitates nucleic acids (both DNA and RNA). RNA contamination can
be removed by ribonuclease (RNase) treatment or lithium chloride (LiCl)
precipitation.
DNA is collected by centrifugation in the form of pellet. Pellet is dissolved in TE buffer
and stored at 4C.

Reagents: Tris buffer, HCl, EDTA, NaOH, SDS, NaCl, β-ME, KoAC, Glacial acetic
acid, Isopropanol, Ethanol

Reagent preparation:
Stock solutions:
1) Tris-HCl (pH 8.0) 1M autoclaved
2) Na2EDTA (pH 8.0) 250 mM autoclaved
3) SDS 10 % -
4) NaCl 5M autoclaved
5) Potassium acetate 5M autoclaved
6) Distilled water - autoclaved
Working solutions:
• Extraction buffer (EB): 50 ml
EB is mixed freshly from stock solutions
Stock Final concentration
Tris-HCl (1 M; pH 8.0) 100 mM

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 Na2EDTA (0.25 M; pH 8.0) 50 mM
NaCl (5 M) 500 mM
-mercaptoethanol 0.25 ml
SDS (10%) 1.25%
Sterile distilled water Rest volume
Warm up to 65° C
• Potassium acetate solution
Composition for 25 ml:
Potassium acetate (5 M) 15 ml
Glacial acetic acid (17.4 N) 2.9 ml
Sterile distilled water 7.1 ml
Store at 4C
• Tris-EDTA (TE) buffer:
Composition for 50 ml:
Stock Final concentration
Tris HCl (1 M; pH 8.0) 10 mM
EDTA (0.25 M; pH 8.0) 1 mM
Sterile Rest volume
Store at 4C
• Isopropanol
• 70 % ethanol

Procedure:
1) The method used was that given by Davis et al. (1980).
2) Two g fresh tender leaves were taken, washed with running tap water and then
with sterile water.
3) Dried the weighed leaves between filter papers.
4) Leaves were homogenized with pestle mortar in 10 ml of prewarmed (65°C in
water bath) extraction buffer and transferred to autoclaved centrifuge tubes.
5) Incubated the tubes at 65ºC for 30 minutes.
6) Added 4 ml of potassium acetate solution. Mixed well with cut pipette tip of 1
ml.
7) Kept the tubes in ice bath for 10 minutes.
8) Centrifuged at 12,000 rpm for 15 minutes at 4°C.
9) Transferred the supernatant to fresh tubes.
10) Added 0.6 v /v isopropanol to each tube and allowed the tubes to lay sidewise
to increase surface area of the content at room temperature (RT) for 10-15
minutes.
11) Centrifuged at 12,000 rpm for minutes at 4° C.
12) Discarded the supernatant.

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13) Washed the pellet with chilled 70 % ethanol twice and centrifuged at 12,000
rpm for minutes at 4° C.
14) The pellet was allowed to completely dry in air.
15) Pellet was dissolved in 100 μl of TE buffer (kept overnight).
16) DNA was visualized by agarose gel electrophoresis as described in next
experiment.

Observation: Pellet of nucleic acids was seen in the tube after precipitation with
isopropanol, which was dissolved in TE buffer.

Result: Isolated nucleic acid was dissolved in TE buffer for visualization by agarose
gel electrophoresis.

Precautions:
• The homogenization should be proper to get good yield of DNA.
• All the reagents should be sterile.
• EDTA should be included in the EB so as to prevent nuclease action, which
would otherwise degrade nucleic acid.
• Prewarmed (65C) buffer should be used and extraction should also be done at
this temperature in order to avoid action of nuclease.
• Phenol chloroform extraction may be included in order to avoid protein
contamination.
• The supernatant after the potassium acetate addition and centrifugation should
be carefully separated so as to avoid any contamination.
• Mixing should be done with cut pipette tip so as to prevent mechanical
shearing or degradation of genomic DNA.
• Mixing should not be vigorous at any stage.
• There should always be proper mixing of all the reagents, but gently.
• Genomic DNA should not be allowed to freeze, i.e., genomic DNA should not
be stored at temperatures lower than 4C.

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 Experiment 9 Commented [SR1]: Paste picture of gel run in lab

Objective: To perform agarose gel electrophoresis for visualization of isolated plant

genomic DNA.

Principle: Electrophoresis is technique of separation of molecules on the basis of

migration of charged particles under the influence of electric field. For separation of
nucleic acids, agarose gel and PAGE (polyacrylamide gel electrophoresis) are used.
Agarose is a polysaccharide isolated from sea weeds (Gelidium and Gracilaria). It
comprises of D-galactose and 3, 6-anhydro L-galactose units linked together by
alternate  (1 → 3) and  (1 → 4) linkages. For normal electrophoresis (quantitative),
the agarose used melts at 95 C and solidifies or gels at 42 C. Besides, there is low
melting /gelation agarose (melting temp. ~65 C, gelation temp. ~25–35 C) also that
is formed by hydroxyethylation is basically used when we are interested in elution of
DNA fragments from the gel.
DNA being negatively charged moves towards anode under electric field. Separation
in agarose gel is based on size and conformation of DNA. The DNA molecules move
through the gel meshwork in globular and extended forms. The largest DNA
molecules move slower than the smaller ones through gel pores. A compact DNA
molecule moves faster through the gel pores. Besides, the gel pore size, which in turn
depends upon agarose concentration, is also an important factor in electrophoresis.
Higher the concentration of agarose used, smaller the pore size and vice versa. Thus,
gels with lower pore size are used for the separation of small molecular weight DNA
molecules. The agarose gel electrophoresis, in general, is efficiently used for the
separation of DNA molecules ranging in size from ~500 bp to ~20 kbp. Above this size
limit, the conventional agarose gel electrophoresis is not used; instead pulsed-field gel
electrophoresis (PFGE) is used.
An agarose gel for the separation of DNA molecules is invariably run as horizontal
submarine or submerge gel, i.e., totally immersed in buffer. The samples are prepared
by mixing bromophenol blue dye for tracking of samples during electrophoresis,
glycerol to increase density of the samples so as to make the samples viscous and
hence easy to load. Ethidium bromide (EtBr) is added either to the samples or in the
gel for visualization of the DNA under U.V. light. EtBr intercalates between the
adjacent bases of DNA and fluoresces (orange color) under U.V. light and hence helps
in visualization of the DNA.

Reagents: Agarose, Tris-Cl buffer (pH 8.), Na2EDTA, Glacial acetic acid, Ethidium
bromide, Bromophenol blue, Glycerol, DNA sample and Distilled water.

Reagent preparation:

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 • 10X TAE buffer (Tris base, acetic acid and EDTA)
Composition for 500 ml:
Tris base 24.2 g
Glacial acetic acid 5.71 ml
EDTA (0.5 M; pH 8.0) 5.00 ml
Distilled water Rest volume
Stored at 4C / RT until required; Final concentration in sample is 1X.
• Loading dye (6X):
Composition for 10 ml:
Bromophenol blue (BPB) 0.25 g (0.25%)
Glycerol 3 ml (30%)
Distilled water Rest volume
Stored at 4°C
• Ethidium bromide:
Stock solution 1 mg / ml
Stored at 4°C
Final concentration in gel or sample 0.5 µg /ml

Procedure:
1) To prepare 50 ml of a 0.8 % agarose solution, measured 0.4 g agarose and
transferred it to a conical flask.
2) Added 50 ml of 1X TAE buffer.
3) Marked the level of buffer on the flask.
4) Melted agarose by boiling in microwave until the solution became clear.
5) Allowed the solution to cool to about ~65 C (tolerable to hands).
6) Poured the above solution into gel casting tray (inbuilt in electrophoresis unit)
fitted with comb to proper level (in order to form wells).
7) It was left undisturbed at room temperature to allow the gel to polymerize.
8) Carefully removed the comb from the polymerized gel.
9) Filled the 1X TAE buffer in the electrophoresis unit up to the level that was
sufficient to submerge the gel along with casting tray.
10) Placed the casting tray along with gel in electrophoresis unit (by slight tilting
in order to avoid entrapment of air bubble underneath the casting tray).
11) Placed the electrodes (cathode and anode) in the electrophoresis unit. Cathode
is placed towards the side of the wells and anode opposite to the wells (so that
the loaded samples move towards anode).
12) Prepared DNA samples by adding 4 l loading dye and EtBr (at a concentration
of 0.5 µg / ml) and mixed well for every 5 l of DNA sample and mixed well.
13) Loaded the prepared samples of DNA in wells (one sample / well).

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14) Connected the electrodes to the power pack at constant volt (50 volts) and
allowed the samples to move under the influence of electric field.
15) The mobility of sample was tracked by the movement of bromophenol blue dye
and electrophoresis was allowed to continue till the tracking dye reached three
fourth of the gel length.
16) Examined the gel under U.V. transilluminator.

Observations: Orange colored bands of high molecular weight plant genomic DNA
were observed under UV illumination. The bands were of high intensity, indicating
high yield.

Result: Plant genomic DNA was isolated in high yields.

Precautions:
• Ethidium bromide is a mutagen and a probable carcinogen and hence should
be handled with care. Avoid spurting of EtBr.
• Gloves should be worn while handling EtBr directly during weighing or
pipetting or handling the gel containing EtBr.
• The work area should not be contaminated with EtBr solution.
• The gel should be discarded at appropriate site meant for its disposal.
• The proper positions of electrodes should be ensured, i.e., anode away from
wells and cathode towards the wells.
• The gel should be properly immersed under buffer.
• Entrapment of bubble underneath the comb or within the gel should be
avoided, as it will affect the flow of electric current.
• U.V. light is damaging and causes burns; hence U.V. light must be used with
caution.
• U.V. light can also affect the eyes; hence avoid exposure of eyes.

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 Experiment 10

Objective:
To estimate RNA content in the given unknown solution by orcinol method

Principle:
This is a general reaction for pentoses and depends on the formation of furfural when
the pentose is heated with concentrated hydrochloric acid. Orcinol reacts with the
furfural in the presence of ferric chloride as a catalyst to give a green colour, which
can be measured at 665 nm.

Reagents:
1) Standard RNA solution: 100 µg/ml
2) Orcinol reagent: Dissolve 0.1g of ferric chloride in 100 ml of concentrated HCl
and add 3.5 ml of 6% w/v orcinol in alcohol

Procedure:
1) Tubes were prepared as indicated in table.
2) The contents of the tubes were mixed well by vortexing / shaking the tubes.
3) Tubes were heated on a boiling water bath for 20 min.
4) The contents of the tubes were cooled.
5) Absorbance was read at 665 nm against reagent blank.
6) The standard curve was plotted by taking concentration of RNA along X-axis
and absorbance at 665 nm along Y-axis.
7) From this standard curve the concentration of RNA in the given sample was
calculated.

Observations:

Tube Volume of Volume Concentration Volume Incubate A665

number standard of of RNA of in
(100 distilled (µg) Orcinol boiling
µg/ml) water reagent water
RNA (ml) (ml) bath
Blank 0.0 1.0 0 2 ml in for 0.00
S1 0.2 0.8 20 each tube 20
S2 0.4 0.6 40 min
S3 0.6 0.4 60 & Cool
S4 0.8 0.2 80
S5 1.0 0.0 100

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 U1 0.5 0.5 -
U2 0.5 0.5 -

Commented [SR2]: Plot standard curve and determine

concentration of unknown RNA. Take absorbance of
unknown as 0.51

Result:
1) The given unknown sample contained ………………… µg RNA/ml. Commented [SR3]: Write that obtained from curve

Precautions:
1) The RNA sample should be analyzed against the reagent blank.
2) The transparent side of the cuvette should be placed in front of the light path.
3) The cuvette should be clean, there should not be any dust or finger impression.
4) The cuvette should be wiped with tissue paper before placing under the light
path in the spectrophotometer.
5) The cuvette should never be held from its transparent side.
6) The DNA sample should be pure.
7) The spectrophotometer should be given proper warm up time to get accurate
readings.
8) The spectrophotometer should be in visible mode to take readings at 595 nm.
9) Mixing of reagents should be done properly.
10) Pipetting should be accurately done.

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