DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE

(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

DEPARTMENT OF BIOTECHNOLOGY

PRACTICAL MANUAL

Core Paper II – Molecular Biology

Course Code :
Class Details : II [Link]. Biotechnology
Semester : IV
Year : 2020 -2021
Course Incharges : Dr. R. Ashwini &
Ms. B. Kiran Sharma
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

CONTENTS

Expt. No Content

1. Isolation and Purification of bacterial genomic DNA

2. Isolation and purification of genomic DNA from plant

3. Isolation and purification of plasmid DNA

4. Isolation and Purification of RNA

5. Determination of Tm of DNA by UV – Vis spectroscopy

6. Estimation of DNA by Diphenylamine method

7. Estimation of RNA by Orcinol method

8. Agarose gel electrophoresis of purified DNA

9. RNA electrophoresis in Formaldehyde gel electrophoresis

10. Gel Documentation

DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

1. Extraction of DNA from bacteria by Phenol/Chloroform Method

Aim:
To isolate Genomic DNA from bacterial culture by using Phenol/Chloroform method.

Introduction:
A phenol-chloroform extraction is a liquid-liquid extraction. A liquid-liquid extraction
is a method that separates mixtures of molecules based on the differential solubilities of the
individual molecules in two different immiscible liquids. Liquid-liquid extractions are widely
used to isolate RNA, DNA, or proteins. The extraction of nucleic acids involves adding an
equal volume of phenol-chloroform to an aqueous solution of lysed cells or homogenized
tissue, mixing the two phases, and allowing the phases to separate by centrifugation.
Centrifugation of the mixture yields two phases: the lower organic phase and the upper aqueous
phase.

Principle:
The organism to be used should be grown in a favorable medium at an optimal
temperature, and should be harvested in late log to early stationary phase for maximum yield.
The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid, etc. Initially
the cell membranes must be disrupted in order to release the DNA in the extraction buffer. SDS
(sodium dodecyl sulphate) is used to disrupt the cell membrane. Once cell is disrupted, the
endogenous nucleases tend to cause extensive hydrolysis. DNA can be protected from
endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a
necessary cofactor for action of most of the nucleases.
Nucleoprotein interactions are disrupted with SDS, phenol or proteinase K. Proteinase
enzyme is used to degrade the proteins in the disrupted cell soup. Phenol and chloroform are
used to denature and separate proteins from DNA. Chloroform is also a protein denaturant,
which stabilizes the rather unstable boundary between an aqueous phase and pure phenol layer.
The denatured proteins form a layer at the interface between the aqueous and the organic phases
which are removed by centrifugation. DNA released from disrupted cells is precipitated by
cold absolute ethanol or isopropanol.

Briefly, the role of each chemical is explained in the table below,

Chemical Role in DNA extraction

It maintains the pH of the solution and also permeabilizes the cell

Tris membrane.

EDTA It is a chelating agent and blocks the activity of DNase enzyme.

DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

It is an anionic detergent which helps in denaturation of cell

SDS membrane protein.

NaCl Prevents the denaturation of DNA

MgCl2 Protects DNA from mixing with other cell organelles

TE buffer It dissolves DNA

Materials and Methods:

• Tris base
• Proteinase K
• Phenol\chloroform (1:1)
• 200 proof ethanol
• RNAase
• Ethanol
• SDS
• EDTA
• Tryptone
• Yeast extract
• NaCl
• LB medium
• TE buffer
• Lysis buffer

Preparation of TE buffer:
• TE buffer (ph 8.0): 10 mm Tris hcl (ph 8.0), 1 mm EDTA (ph 8.0)
• 10% SDS: Dissolve 10 g of SDS in 100 ml autoclaved distilled water.
• Proteinase K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water.
• Phenol – Chloroform Mixture: Prepare Phenol: Chloroform mixture in 1:1 ratio
• 5M Sodium Acetate: Dissolve 41 g of sodium acetate in 100 ml distilled water and
adjust ph with dilute acetic acid (ph 5.2).
• Isopropanol
• 70% Ethanol
Procedure:
1. 2 ml overnight culture is taken and the cells are harvested by centrifugation for 10
minutes
2. 900 µl of TE buffer is added to the cell pellet and the cells are resuspended in the buffer
by gentle mixing.
3. 100 µl of 10% SDS and 5 µl of Proteinase K are added to the cells.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

4. The above mixture is mixed well and incubated at 37º C for an hour in an incubator.
5. 1 ml of phenol-chloroform mixture is added to the contents, mixed well by inverting
and incubated at room temperature for 5 minutes.
6. The contents are centrifuged at 10,000 rpm for 10 minutes at 4º C.
7. The highly viscous jelly like supernatant is collected using cut tips and is transferred to
a fresh tube.
8. The process is repeated once again with phenol-chloroform mixture and the supernatant
is collected in a fresh tube.
9. 100 µl of 5M sodium acetate is added to the contents and is mixed gently.
10. 2 ml of isopropanol is added and mixed gently by inversion till white strands of DNA
precipitates out.
11. The contents are centrifuged at 5,000 rpm for 10 minutes.
12. The supernatant is removed and 1ml 70% ethanol is added.
13. The above contents are centrifuged at 5,000 rpm for 10 minutes.
14. After air drying for 5 minutes 200 µl of TE buffer or distilled water is added.
15. 10 µl of DNA sample is taken and is diluted to 1 or 2 ml with distilled water.
16. The concentration of DNA is determined using a spectrophotometer at 260/280 nm.
17. The remaining samples are stored for further experiments.

Result:
Bands were seen in 1% of agarose gel under UV which confirmed the presence of DNA.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

2. Plant Genomic DNA Extraction using CTAB

Introduction:
The search for a more efficient means of extracting DNA of both higher quality and
yield has lead to the development of a variety of protocols, however the fundamentals of DNA
extraction remains the same. DNA must be purified from cellular material in a manner that
prevents degradation. Because of this, even crude extraction procedures can still be adopted to
prepare a sufficient amount of DNA to allow for multiple end uses. DNA extraction from plant
tissue can vary depending on the material used. Essentially any mechanical means of breaking
down the cell wall and membranes to allow access to nuclear material, without its degradation
is required. For this, usually an initial grinding stage with liquid nitrogen is employed to break
down cell wall material and allow access to DNA while harmful cellular enzymes and
chemicals remain inactivated. Once the tissue has been sufficiently ground, it can then be
resuspended in a suitable buffer, such as CTAB. In order to purify DNA, insoluble particulates
are removed through centrifugation while soluble proteins and other material are separated
through mixing with chloroform and centrifugation. DNA must then be precipitated from the
aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is
then resuspended and stored in TE buffer or sterile distilled water. This method has been shown
to give intact genomic DNA from plant tissue. To check the quality of the extracted DNA, a
sample is run on an agarose gel, stained with ethidium bromide, and visualised under UV light.

Materials:
• CTAB buffer
• Microfuge tubes
• Mortar and Pestle
• Liquid Nitrogen
• Microfuge
• Absolute Ethanol (ice cold)
• 70 % Ethanol (ice cold)
• 7.5 M Ammonium Acetate
• 55o C water bath
• Chloroform : Iso Amyl Alcohol (24:1)
• Water (sterile) Agarose
• 6x Loading Buffer
• 1x TBE solution
• Agarose gel electrophoresis system
• Ethidium Bromide solution
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

Reagent Preparation:

CTAB buffer 100ml

2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)
10.0 ml 1 M Tris pH 8.0
4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt)
28.0 ml 5 M NaCl
40.0 ml H2O
1g PVP 40
Adjust all to pH 5.0 with HCL and make up to 100 ml with H2O.

1 M Tris pH 8.0
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of
concentrated HCL. Allow the solution to cool to room temperature before making the final
adjustments to the pH. Adjust the volume to 1 L with H2O. Sterilize using an autoclave.

5x TBE buffer
54 g Tris base 27.5 g boric acid 20 ml of 0.5M EDTA (pH 8.0) Make up to 1L with water. To
make a 0.5x working solution, do a 1:10 dilution of the concentrated stock.

1% Agarose gel
1 g Agarose dissolved in 100 ml TBE

Procedure:
1. DNA was isolated from the fresh leaf samples using the modified CTAB method.
2. Around 0.2g of the leaves was ground with preheated extraction buffer (3 M NaCl, 25
mM EDTA 100mM Iris HC1, 0.2% ß-mercaptoethanol, 2% PVP,3% CTAB) and it was
incubatedat 60°C for 30 min.
3. The homogenate was extracted with chloroform and isoamylalcohol (24:1) at 10,000
rpm for 15 min at 4°C.
4. The upper phase was extracted with equal volume of phenol: chloroform:
isoamylalcohol ([Link]) at 10,000 rpm for 10 min at 4°C.
5. The aqueous layer was again extracted with chloroform: isoamylalcohol (24:1).
6. The DNA in the aqueous layer was precipitated by adding equal volume of ice-cold
isopropanol and incubated at20°C for an hour.
7. The precipitate was centrifuged at 12,000 rpm for 15 min.
8. DNA pellet obtained was washed with 70% ethanol and air dried.
9. The pellet was finally resuspended in TE buffer (Jayaram et al., 2009).
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

DNA quality confirmation:

1. Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE
buffer in a microwave for approximately 2 min. Allow to cool for a couple of minutes
then add 2.5 μl of ethidium bromide, stir to mix.
2. Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20
min at room temperature on a flat surface.
3. Load the following into separate wells o 10 μL 1kb ladder o 5 μL sample + 5 μL water
+ 2 μL 6x Loading Buffer
4. Run the gel for 30 min at 100 V
5. Expose the gel to UV light and photograph (demonstration)
6. Confirm DNA quality, presence of a highly resolved high molecular weight band
indicates good quality DNA, presence of a smeared band indicates DNA degredation.

Result:
Bands were seen in 1% of agarose gel under UV which confirmed the presence of DNA.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

3. Isolation and purification of Plasmids

Aim:
Isolation of plasmid DNA from E. coli by alkaline lysis method.

Principle:
The basis of the technique is that there is narrow pH range at which non supercoiled
DNA is denatured where as supercoiled plasmids are not. If sodium hydroxide is added to the
cell extract so that the pH is adjusted to 12.0 to 12.5, then the hydrogen bonding is non
supercoiled. DNA molecules will be broken, causing the double helix to unwind and two
polynucleotide chain to separate. If acid is now added these denatured bacterial DNA sequence
will re-aggregate into a tangled mass. Centrifugation will pellet the insoluble network leaving
pure plasmid DNA in the supernatant. Addition of isopropanol further precipitate the plasmid
DNA.

Materials Required:
• [Link] straining containing plasmid
• Glucose
• Tris chlorid
• EDTA
• Sodium hypochloride
• SDS
• Glacial acetic acid
• Isopropanol
• Absolute alcohol
• Conical flask
• Cotton plugs
• Aluminum foil
• Centrifuge
• Paper towel
• Ice flasks

Preparation of Reagents:
Solution I
• Glucose 50ml
• Tris chloride 50mM
• EDTA 10mM
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

Solution II
• NaOH 0.2N
• SDS 1%

Solution III
• Potassium acetate (5m) 60ml
• Glacial acetic acid 11.5ml
• Autoclave water 28.5ml
• 75% of ethanol

Procedure:
1. 100 ml of autoclave lb media was taken in conical flask of 250 ml.
2. To the media was added 100ml of inoculum.
3. Culture was incubated at 37C over-night in a shaker at 70 revolutions per minute.
4. 1.5 ml of the bacterial culture was taken in a 22 ml Eppendorf tube
5. Harvesting of the bacteria was done by subject in the culture at 3000 rpm for 10 minutes
at 4C.
6. Supernatant was removed and then pellet was collected.
7. To the pellet 30μl of solution one it was then mixed nicely by vertexing and mixing
with pipette.
8. Tube was taken from ice and to it added 60μl of freshly prepared solution II. It was
mixed gently and kept at room temperature.
9. To the same tube was added 45μl of solution II. It was then kept in ice for 10mins.
10. Centrifuge the bacterial lysate for 30 mins at 8000-12000 rpm
11. Carefully decant the supernatant.
12. Added 0.6 volume of isopropanol. Mix it and then keep it at room temperature for an
hour.
13. Recover the plasmid by centrifugation at 3000 rpm for 30mins.
14. Decant the supernatant.
15. Collect the pellet.
16. Wash the pellet with 75% of ethanol.
17. Dry the pellet by keeping it in dessicator or drying it under fan.
18. Dissolve it in 20μl of autoclave triple distilled water.
19. Run it on 1% of agarose gel.

Results:
Bands were seen in 1% of agarose gel under UV which confirmed the plasmid DNA.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

4. Isolation and purification of RNA

Aim:
To isolate Genomic DNA from bacterial culture by using Phenol/Chloroform method.

Principle:
Total RNA is isolated and separated from DNA and protein after extraction with a
solution called as Trizol (Sigma). Trizol is an acidic solution containing guanidinium
thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases.
This is followed by centrifugation. Under acidic conditions, total RNA remains in the upper
aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower
organic phase. Total RNA is then recovered by precipitation with isopropanol. RNase enzymes
can be inactivated by including diethyl pyrrocarbonate (DEPC).

Materials required:
• Bacterial Culture
• Trizol
• Chloroform
• Isopropanol Solution
• TAE Buffer
• 70% Ethanol

Protocol:

1. Take 800 Μl Of Bacterial Culture In A Fresh Eppendorf.

2. To this add 160 μl of trizol (1/5th of culture volume).
3. The solution was mixed well by pipetting several times.
4. To this add 32 μl of chloroform (1/5th volume of trizol).
5. Incubate for 2 to 5 minutes and centrifuge at 12000 rpm for 15 minutes at 4° c
6. Transfer the aqueous phase into a new tube and add equal volume of isopropanol. Mix
well.
7. Centrifuge at 10000 rpm for 10 minutes at 4° c.
8. Discard the supernatant and resuspend the pellet in 70% ethanol.
9. Again centrifuge at 10000 rpm for 10 minutes at 4° c.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

10. Discard the supernatant. Air dry the pellet at 37° c for 10-15 minutes.
11. Resuspend the pellet in 50 μl of te buffer.
12. Analyze the RNA sample quantitatively and qualitatively.

Results:
RNA was isolated and analyzed by __________
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

5. Melting temperature of the double stranded DNA

Aim:
To determine the melting temperature of given double stranded DNA solution.

Principle:
Melting temperature is the temperature at 50% of the DNA has undergone denaturation.
When a double stranded DNA solution is heated two strands gets separated due to disruption
of hydrogen bonds between complementary basis. Such DNA is known as denaturated DNA
and the process is known as denaturation. If above solution is allowed to cool at room
temperature, complementary strands resembled to form duplex reannealed DNA. Upon
denaturation, the absorbance of DNA at200nm increases by 30-40% due to exposure of bases.
This effect is caused by hyper chromic effect. A curve between temperature and absorbance as
platted. Temperature corresponding to the midpoint of the curve is called the melting
temperature of mDNA and it donate the temperature at which 50% of DNA undergone
denaturation.
If DNA sample is used is double stranded, a sharp increase in optical density
(absorbance at 260nm) absorbed in between 60-80°c during denaturation. Tm is largely
dependent on (G+C) content of DNA and if higher the (G+C) content, higher the Tm (Melting
temperature).
% (G+C) = (Tm-69.3) x 2.44

Materials required:
• DNA
• Test Tube
• Water bath
• Spectrophotometer

Procedure:
1. Switch on the spectrometer.
2. Allow sufficient period for warming up and set it to 0absorbance at 260nm with
autoclave water.
3. Measure A260 of DNA sample at 25c, 40c, 60c, 70c, 80c respectively.
4. Distribute above solution into 5 test tubes and put each test tube in a water bath
maintained at different temperature.
5. Allow the tube to stand for 15minutes. After incubation quickly cool all the test tubes
except the tube at 25c.
6. By placing them in the ice bath for annealing which occurs only if the sample is cool,
but reannealing is negligible if cooling is instantaneous.
7. Record the absorbance at A260nm.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

8. Calculate the absorbance A260 at 25c for each of the following temperature, 25c, 40,
60c, 70c, 80c and plot absorbance rate against the temperature.
9. Determine midpoint of increase in absorbance and by extrapolation. Find
corresponding temperature will represent Tm for DNA sample.
10. Calculate the % (G+C) content of DNA using the equation.
%(G+C) = (Tm – 69.3)x2.44

Result:
The maximum absorbance of DNA is at 260nm which is ____________

Observation:
S. No. Incubation temperature OD At 260 nm
1. 25 ˚C
2. 40 ˚C
3. 60 ˚C
4. 70 ˚C
5. 80 ˚C

Calculation:
Maximum absorbance of DNA = ____ at 260nm
Melting temperature =____
% (G+C) = (Tm-69.3)x2.44
% (G+C) =
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

6. Estimation of DNA by Diphenyl amine method

To be written on the right side of the observation:

Aim:

To estimate the amount of DNA present in the given unknown solution by Diphenyl amine
(DPA) method.

Principle:

Nucleic acid is usually present in combination with protein called as nucleoprotein. During
isolation this complex is broken down and the nucleic acid is treated with alcohol. The Deoxy ribose in
DNA undergoes dehydration in the presence of acid to form hydroxilevulinic aldehyde which reacts
with Diphenylamine to produce a blue coloured condensation product with absorption maximum at 590
nm.

Reagents required:

i) Stock standard solution:

100 mg of DNA was weighed accurately and dissolved in Saline Sodium Citrate (SSC) and
made upto 100 ml with the same in a standard flask.

Concentration = 1 mg/ml

ii) Working standard solution:

10 ml of stock solution was made upto 100 ml with saline sodium citrate in a standard flask

Concentration = 100 μg /ml

iii) Diphenyl amine reagent:

5 g of Diphenyl amine was mixed in 500 ml of acetic acid containing 12.5 ml of concentrated
sulphuric acid.

iv) Saline Sodium Citrate (pH 7.4):

0.14 M (Molar) Sodium chloride containing 0.02 M trisodium citrate was prepared.

v) Reagent:

2 M Sodium chloride – 11.68 g of NaCl was dissolved in 100 ml of distilled water.

Procedure:

0.5 – 2.5 ml of standard DNA solution in the concentration range of 50 – 250 mg was pipetted
out into test tubes labelled as S1 – S5. The given unknown solution was made upto 100 ml with saline
sodium citrate in a standard flask. 0.5 ml and 1.0 ml of the unknown solution was pipetted into test
tubes labelled as U1 and U2. The volume in all the test tubes was made upto 3 ml with Saline Sodium
Citrate. 3 ml of Saline Sodium Citrate alone serves as blank. 5 ml of Diphenly amine reagent was added
to all test tubes and the contents were mixed gently. The tubes were kept in boiling water bath for 10
mins, cooled and the intensity of blue colour developed was read colorimetrically at 590 nm.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

A standard graph was plotted by taking the concentration of DNA in X axis corresponding to
the absorbance values on Y axis. From the graph, the amount of DNA present in the given unknown
solution was calculated.

Result:

The amount of DNA present in the given unknown solution was found to be ____ mg.

To be written on the Left side of the observation:

This should be in one page and the tabular column with calculation should
be in another page.
Stock Standard Solution:

100 ml of DNA was weighed and dissolved in Saline Sodium Citrate and made upto 100 ml
with the same in a standard flask.

Concentration = 1 mg/ml

Working Standard Solution:

10 ml of stock standard solution was made upto 100 ml with Saline Sodium Citrate in a standard
flask.

Concentration = 100 μg /ml

Estimation of DNA:

S. Particulars B S1 S2 S3 S4 S5 U1 U2
No.
1. Volume of working standard DNA solution - 0.5 1.0 1.5 2.0 2.5 - -
(ml)
2. Concentration of protein in standard DNA - 50 100 150 200 250 - -
solution (g)
3. Volume of unknown solution (ml) - - - - - - 0.5 1.0

4. Volume of Saline Sodium Citrate buffer (ml) 3.0 2.5 2.0 1.5 1.0 0.5 2.5 2.0

5. Volume of Diphenyl amine reagent (ml) 5.0

Tubes were mixed well and kept in boiling water

bath for 10 mins and cooled
6. Absorption at 540 nm

Calculations:

i) Unknown solution:

0.5 ml of unknown DNA solution corresponds to _____ OD

DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

_____ OD of unknown DNA solution corresponds to _____ mg of DNA.

0.5 ml of unknown DNA solution contains _____ mg of DNA.

Therefore, 100 ml of the given unknown DNA solution contains

= _____ x 100
0.5
= ______ μg

= ______ mg
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

7. Estimation of RNA by Orcinol method

( To be written on the right side of the observation )

Aim :
To estimate the amount of RNA present in the given unknown solution.
Principle :
The RNA was estimated colorimetrically using acid orcinol reagent. Acid hydrolysis of
RNA releases the ribose which in presence of strong acid undergoes dehydration to yield
furfural. Orcinol reacts with furfural in the presence of ferric chloride to yield green colour.

Reagents required :
1. Stock standard RNA solution
100mg of RNA was weighed end dissolved in 100ml of ice cold saline sodium
citrate in a standard flask .
Concentration = 1 mg / ml

2. Working standard solution

10ml of the stock was diluted to 100ml using distilled water in a standard flask .
Concentration = 100 µg/ml

3. Orcinol acid reagent

100mg of ferric chloride is dissolved in 100ml of concentrated hydrochloric
acid and then 3.5ml of 6% solution of alcohol orcinol reagent is added .

4. Saline Sodium Citrate Buffer 0.14 M pH 7.4

0.14M Sodium chloride containing 0.02M trisodium citrate.
5. 6% Alcohol Orcinol reagent

Procedure :
Estimation of RNA:
0.5 – 2.5 ml of standard RNA Solution with a concentration range of 50-250 μg was
pipetted out into tubes labelled as S1 – S5 . 0.5ml and 1.0 ml of the unknown RNA solution
was pipette out into tubes labelled as U1 and U2 . The volume in all the tubes were made upto
3ml with saline sodium citrate buffer . 3ml of saline sodium citrate alone serves as blank . 3ml
of orcinol reagent was added to all the tubes and the contents were mixed well. The tubes were
kept in boiling water bath for 15minutes, cooled and intensity of green colour developed was
read colorimetrically at 660nm.
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

A standard graph was plotted by taking the concentration of RNA in X- axis and corresponding
absorbance on Y-axis. From the standard graph the amount of RNA present in the given yeast
extract was calculated.
Result:
The amount of RNA present in 100ml of unknown solution was found to be ________ of
RNA
( To be written on the left side of the observation )
1. Stock standard RNA solution
100mg of RNA was weighed end dissolved in 100ml of ice cold saline sodium
citrate in a standard flask .
Concentration = 1 mg / ml

2. Working standard solution

10ml of the stock was diluted to 100ml using distilled water in a standard flask .
Concentration = 100 µg/ml

Estimation of RNA
S. Particulars B S1 S2 S3 S4 S5 U1 U2
No.
1. Volume of working standard RNA - 0.5 1.0 1.5 2.0 2.5 - -
solution (ml)
2. Concentration of protein in standard - 50 100 150 200 250 - -
RNA solution (g)
3. Volume of unknown solution (ml) - - - - - - 0.5 1.0

4. Volume of Saline Sodium Citrate 3.0 2.5 2.0 1.5 1.0 0.5 2.5 2.0
buffer (ml)
5. Volume of Orcinol reagent (ml) 5.0

Tubes were mixed well and kept in boiling water

bath for 15 minutes
6. Absorption at 660 nm

Calculation :
Unknown
0.5 ml of unknown RNA solution corresponds to _______ OD
_______ OD corresponds to _____ μg of RNA
0.5 ml of unknown solution RNA contains ______ μg of RNA
Therefore, 100ml of unknown solution contains × 100
0.5
= ________μg
=______ mg of RNA
DWARAKA DOSS GOVERDHAN DOSS VAISHNAV COLLEGE
(Autonomous)
College with Potential for Excellence, Linguistic Minority Institution
Affiliated to University of Madras
Arumbakkam, Chennai – 600 106

8. Agarose gel electrophoresis

Aim:
To separate , analyse and quantitate nucleic acids.

Principle:
Agar , which is obtained mainly from the seaweed Gelidium Species(a rea algae) is a
powdery mixture composed of 2-galactose based polymer viz agarose and agarose pectin. Agar
, gels which boiled in a aqueous buffer and then cooked at 40°C. Agarose gels have a high
water content, good fibre structure , large pore size , and low friction resistance . However high
sulfate content of agar gel causes severe electroendosmosis . Therefore, non sulphated agarose
component of agar is purified and agarose is new being used extensively for electrophoretic
separation of nucleic acid.
Agarose has an average molecular weight of 12000 and contain about 35-40 agarose base
unit. Even the purified neutral fraction of agarose contains some charge group consisting of L-
galactose 6 sulphate moieties. Low charge agarose is found to be suitable for electrophoresis
of nucleic acid.
The core links are held together by hydrogen and hydrophobic bonding. By changing the
gel concentration, the pore size can be altered ie. higher the concentration of agarose , smaller
the pore size and vice versa. Because of larger pore size , even at lower concentration , agarose
gel are widely used for separation of DNAs and RNAs but not the protein.

Figure 1: Structure of Agarose

Materials Required:
• Submarine electrophoresis unit
• Power pack
• UV trans illuminator
• Distilled water
• Agarose solution
• TBE , TAE Buffer
• Glass wares, conical flask ,etc.
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Procedure:
1. Prepare 20ml of 1% agarose solution in the buffer provided (0.5× TBE buffer)
2. Boil the agarose until it completely dissolve and make sure no obvious particle of
agarose remain in the suspension.
3. When the gel temperature is around 40°C , add 2µl of Ethidium Bromide and mix
properly.
4. Seal the gel casting tray on both sides and place the comb on the gel tray in
appropriate place.
5. Power the agarose mixture into tray containing comb
6. After complete solidification of agarose remove the seal from either sides of the
tray, without disturbing the gel.
7. Keep the gel tray in the tank containing 0.5× TBE buffer with the wells in the
cathode side(negative)
8. The buffer level is maintained above the gel tray .
9. Gently lift the comb without damaging the well, and now it is ready for loading.
10. Connect the power works between the electrophoresis tank and power pack before
loading the sample.
11. Prepare sample for electrophoresis by adding 5µl gel loading dye into ligated
sample and mix cells by pipetting.
12. Load 20µl of sample to each well.
13. For controlled (unligated) aliquot 3µl of lamdaƛ hierel III digest(from the vial
provided) into the microphage tube, with that add 3µl of gel loading dye mix it and
load (6µl)to the ready well.
14. After loading switch on the power bank and adjust the voltage 50v and/or 100v.
15. Continue until the dye reaches 1/3rd or the above.

TO BE WRITTEN ON LEFT SIDE

TBE buffer
TRIS BORATE (1× TBE) FOD 10×BUFFER/LIT
8.9 mµ TBE 108.00g
8.9 mµ boric acid 55.00ml
2mµ Na2 EDTA 7.44g

TAE buffer
TRIS ACETATE(1×TAE) FOD 50×BUFFER/LIT
50mµ tris 302.50g
25mµ glacial CH3COOH 71.40ml
1mµ Na2 EDTA 18.60g
pH 8.3
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Sample loading dye:

• Prepare by mixing 0.7ml of 10% glycerol or 0.2ml of 60%sucrose in o.2ml 10×TBE
bugger or 50× TAE buffer and o.1 ml of 3% Bromophenol Blue solution,
• Mix 1.5ml of sample loading dye with 10µl of DNA sample and load.

Agarose solution:
Distilled water 30ml
50×TAE 0.6ml
Agarose (0.8) 0.24g

Result:
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9. Formaldehyde gel electrophoresis of total RNA

Introduction:
RNA has the tendency to form both secondary and tertiary structures that can impede
its separation by electrophoresis. As such , identical species of RNA exhibiting varying degrees
of intramolecular base-pairing,migrate at different rate and result in the smearing of distinct
RNA molecules. Consequently, the electrophoresis of RNA needs to be performed under
denaturing conditions. Heat denaturing the RNA sample prior tpelectrophoresis is insufficient,
as secondary structures will simply reform unless a denaturing system is used. Successful
electrophoresis of RNA is therefore accomplished in two steps: RNA is heat denatured prior to
electrophoresis; and during electrophoresis, conditions are established that maintain the RNA
in a denature state.
The methodology described in this chapter involves the use of formaldehyde as a
denaturant within agarose gel. In addition, both formaldehyde and formamide are added to the
sample befor electrophoresis to aid the denaturation of the RNA sample. For procedures such
as northern analysis, in which RNA is transferred or blotted from the gel to a solid matrix for
subsequent hybridisation, the optimal balance between electrophoretic resolution and
efficiency of transfer is achieved with a 1-1.2% agarose gel (see refs.1-3).
Materials Required:
1. Formaldehyde
2. Formamide
3. RNase-free water
4. 10X MOPS buffer
5. Agarose
6. Ethidium bromide solution (10mg/ml)
7. Horizontal electrophoresis tank and a 11x14 cm casting tray
8. RNA-loading buffer

TO BE WRITTEN ON THE LEFT SIDE

Role of Materials used:
Formaldehyde Formaldehyde is a suspected nose, nasopharynx, and liver carcinogen; it is
toxic both through inhalation and ingestion. Its use should therefore be
restricted to a ventilated fumehood. Formaldehyde is routinely supplied as a
37%(v/v) stock solution. It should be stored at room temperature and out of
direct sunlight to prevent oxidation.
Formamide The use of this chemical should be restricted to the fumehood, as it is a
suspected teratogen and is irritating to the eyes, skin, and respiratory system.
Formamide should be stored in the dark and at room temperature to prevent
oxidation.
RNase-free This can be achieved by treating the water first with diethyl pyrocarbonate
water (DEPC). Add DEPC to water to a final concentration of 0.1%. Incubate
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overnight at 37 degree celcius and autoclave to destroy any residual DEPC.

DEPC is an efficient, nonspecific inhibitor of RNase, however it is
carcinogenic and should be handled in a fume hood with extreme care.
10x MOPS (0 2 =M MOPS (3-(N-Morphilino)propanesulfonic acid) pH 7.0; 50mM
buffer sodium acetate, pH 6.0, 10mM EDTA, Ph 8.0. this solution is prepared using
RNase-free water. When autoclaved it assumes a characteristic golden color
and can be stored at room temperature
Agarose Store at room temperature
(molecular
biology-grade)
Ethidium This is a powerful mutagen, extreme care must be taken when using this
bromide substance. Store at 4 degree celcius.
solution
(10mg/ml)
RNS- loading 50% glycerol, 1mM EDTA and 0.4% bromophenol blue, made up to the
buffer required volume with DEPC-treated water.

Procedure:
1. Denature the RNA in a sterile RNase-free microcentrifuge tube by mixing the
following:
● 10 µg of RNA ( in a final volume of 5pl with DEPC-treated water),
● 2 µl of 10x MOPS solution,
● 3.5 µl of formaldehyde,
● 10 µl of formamide. Incubate the RNA solution at 65ºC for 15 min in a fumehood
and transfer immediately to ice.
2. For a 1% agarose gel using an 11x14 cm casting tray, mix 0.8g of agarose with 57.5ml
of sterile RNase-free water and boil to dissolve agarose. Cool to 60ºC, then add 8ml of
10x MOPS buffer, 14.5ml of 37% (12.3M) formaldehyde and finally 8 µl of ethidium
bromide solution ( at a concentration of 10mg/ml). mix the compenents of the gel by
gently rotating the bottle, being careful not to introduce any air bubbles. Pour the gel
into the prepared casting tray (in a fumehood), insert the comb and let it set for atleast
60min at room temperature.
3. After cooling the denatured RNA solution in ice, add 2 µl of sterile RNA loading buffer.
4. Fill the buffer reservoirs and cover the gel with 1x MOPS buffer.
5. Pre-electrphorese the gel at 50 V for 10 min.
6. Load the heat denatured samples into the wells and run the gel at 50 V until the
bromophenol blue has moved approximately half to three quarters of the way along the
gel ( see note 1).
7. Visualize RNA using a 254nm short wave ultra-violet transilluminator (see noter2-7).
Gel can be photographed using a polaroid land camera with 66S polaroid film.

Result:
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10. Gel Doc Experiment

Aim:
To provide instruction for the proper use of the gel documentation system (Gel Doc)
Procedure:
To move a gel from one room to another in the same wing it is recommended that the gel
be placed in a secondary container (i.e. small tray) so it can be transported without need for
gloves. It is also permissible to use the ‘one-glove’ technique described below:
1. Remove the glove from your dominant hand. Use the ungloved hand to open all doors,
and carry the gel in the gloved hand
2. Open the door to the exposure chamber with the ungloved hand. Place the gel into the
exposure chamber using the gloved hand and a spatula.
3. Use the ungloved hand to manipulate all controls on the Gel Doc system.
4. Sign the log book with the ungloved hand.
5. When the documentation process has finished, open the chamber door with the
ungloved hand, and remove the gel with the gloved hand and a spatula.
6. Using the gloved hand, use a tissue to wipe the glass surfaces of the Gel Doc.
7. Use the gloved hand to carry the gel back to your laboratory, opening all doors with the
ungloved hand.

Starting the Program:

8. Ensuring you are using an ungloved hand, click the mouse to activate the monitor. Open
the Gel Doc software if it is not already open.
9. On the menu bar, select ‘File’, cursor down to ‘Acquire’ and select ‘Gel Doc’.

Setting up the gel:

10. Open the chamber door with the ungloved hand and load the gel into the chamber with
the gloved hand. Centre the gel, using the monitor to assist in visualization.
11. Close the door and switch on UV Light.
12. With an Ungloved hand, adjust the focus, zoom and aperture on the camera to obtain
the optimal image.
13. In the Gel Doc Window, click ‘Capture’. Select the hatched-box icon in this window
and drag it to select the area of interest.
14. On the menu bar, select ‘Edit’, and cursor down to ‘Extract’. A new window will appear
with the final picture. You may wish to adjust the image properties (brightness/contrast)
at this point.

Printing:
15. On the menu bar, select ‘File’, cursor down to ‘Video Print’ and click to print.
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16. If the roll has a pink stripe, after your image has printed, obtain a new roll from the
stock room and install as per the directions on the printer.
17. Close the Windows containing the extracted and original images. Click ‘Don’t Save’ in
the pop-up dialog box.
18. Closing the Program:
19. Turn off the UV light.
20. With a gloved hand, remove your gel from the chamber and wipe down the surfaces
with a tissue or paper towel.
21. Close the door to the chamber with a clean hand.
22. Record your name and the number of photos taken in the log book.

Figure 1: GEL DOC SYSTEM

Figure 2: WORKING OF GEL DOC

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Precautions:
• All operators must receive training prior to using the equipment. Training may be
delegated to a qualified individual, but it remains the responsibility of the supervisor to
ensure their personnel are adequately trained.
• Ethidium Bromide is a known mutagen. Always wear a lab coat and gloves when
handling ethidium bromide solutions and stained agarose gels.
• Do not look into the UV light source without face or eye protection.

Result: