Experiment -1

Objective:
Extraction of genomic DNA from plant.
Principle:
All plants contain DNA as their genetic material. In Eukaryotic plants DNA is present inside
the nucleus. Plants cells are surrounded by hard cell wall, which is to be broken to extract
DNA. After breaking of cell wall, contents of cell are released and DNA is present in
nucleus. This nucleus is lysed using detergents. Detergent dissolves nuclear membrane and
DNA is released. This DNA is further purified from protein, lipids and other contaminating
material and then precipipated using isopropanol.
Requirements:
Chemicals Plastic and Glasswares
CTAB (Cetyl Trimethyl Ammonium Morter and pestle
bromide) or SDS (Sodium Dodecyl sulphate) Pipettes
Isopropanol sterile tips
Tris microcentrifuge tubes
EDTA Sterile glassware
Distill water Water bath
Liquid nitrogen centrifuge
Phenol
Chloroform
Isoamyl alcohol
Ethanol

Buffer and solutions to be prepared

1. Extraction(CTAB) Buffer
• 1.4 M Na Cl 100 mM Tris (pH 8.0)
• 20 mM EDTA (pH 8.0)
• 2% Beta-Mercaptoethanol
• 2% CTAB
2. Isopropanol
3. Saturated phenol pH 8.0
4. Chloroform : isoamylalcohol ( 24:1) mixture
5. Tris:EDTA ( 10mM:1mM) pH 8.0 10 mM Tris
6. RNase A (10mg / ml): Dissolve RNase A in 10mM Tris-Cl, pH 7.5, 15 mM NaCl. Heat
at 1000 C for 15 min. Cool to room temperature. Store as aliquots at -20°C.
7. 70% ethanol

Procedure:
1. Weigh 2 g of clean young leaf tissue and grind to fine powder with a pestle and
mortar after freezing in liquid nitrogen.
2. Transfer grinded leaves powder into centrifuge tube and add 10 ml extraction buffer
to it, mix it vigorously and keep at 65°C in a water bath for one hour.
3. After Incubate of one hours at 65°C for one hour, allow the tube to cool and come to
room temperature.
4. Add 10 ml of ‘chloroform : isoamyl alcohol’ and mix it gently.
5. Centrifuge at 10,000 rpm for 10 min at 25°C.
6. After centrifugation transfer upper aqueous phase to a fresh centrifuge tube.
 7. Add 0.6 volume of chilled isopropanol to aqueous phase and let the DNA to
precipitate for 30 minutes, by keeping it in – 20°C deep freezer.
8. Spool out the DNA and discard the excess of buffer.
9. Add 0.5 ml of 70% ethanol to isolated DNA, mix gently and keep at room
temperature for 15 minutes. After 15 minutes decant excess of alcohol.
10. Repeat step 9 for 2 more times and let the DNA air dry.
11. Dissolve DNA in 1 ml of 10: 1 TE buffer.
12. Add RNAse A (10 microlitrel) and incubate at 37°C for one hour.
13. Add equal volume of phenol : chloroform : isoamyl alcohol (25:24:1), mix properly
for at least 5 min and centrifuge at 10000 rpm for 10 minutes.
14. Precipitate DNA by adding 1/10 volume of 3M NaCl solution and 2.0 times of the
total volume chilled ethanol. Mix gently, let DNA precipitate and spool out DNA.
15. Air dry the DNA pellet and store at – 20°C .

Observation and result:

After adding isopropanol or chilled alcohol DNA is precipitated out at light white threads,
which can be further checked with spectrophotometer.

Experiment-2
Objective:
Qualitative estimation of DNA by Diphenylamine (DPA) Test.
Principle
When DNA is treated with diphenylamine under acidic conditions, a blue coloured
compound is formed which give absorbance at 595 nm. This reaction is given by all 2-deoxy
pentose sugars and not specific to DNA only.
In acidic solution deoxypentose is converted to b-hydroxy lerulin aldehyde, which
reacts with diphenylamine to produce a blue coloured complex. In DNA,
only purine nucleotide are responsible for this reaction hence the value given by this reaction
represent half of the DNA present.
Requirements:
1. Purified DNA sample.
2. Saline solution of Solution of sodium chloride and sodium citrate
3. Diphenyl amine reagent: Dissolved in 10 gms of pure diphenyl amine in 1
liter of glacial acetic acid and add 25 mL of
concentrated H2SO4. This solution must be prepared fresh. Boiling water bath
Procedure
1. Dissolve given DNA sample in 5 ml of saline solution.
2. Take 2 ml of DNA solution and add 2 ml of DPA reagent to it.
3. Heat on a boiling water bath for 10 minutes.
Result:
After 10 minutes blue colour compound appears, which indicates the presence of DNA in the
sample.
Conclusion:
Formation of blue colour is due to reaction of DPA with 2-deoxy ribose sugars. In DNA
deoxyribose sugars are present hence blue colour in the sample indicates presence of DNA in
it.
 Experiment -3
Objective:
Qualitative estimation of DNA by Killer-Killani test.
Principle:
When deoxy-sugars react with ferric chloride under acidic conditions, a blue-brown ring is
formed at the junction of acid and ferric chloride in presence of deoxy-sugars. DNA is made
up of deoxy-sugars, hence it give positive results for this test.
Requirements
1. Purified DNA sample.
2. 1% ferric chloride solution
3. Glacial acetic acid
4. Concentrated sulphuric acid.
Procedure:
1. Dissolve given DNA sample in 5 ml of saline solution and add glacial acetic to acid,
to acidify the mixture.
2. Add one drop of 1% ferric chloride solution to it and mix it well.
3. Add conc. Sulphuric acid from the side wall of test-tube.
Result:
A blue-brown color ring form at the junction of sulphuric acid and ferric chloride solution.
Conclusion:
Formation of blue-brown ring at the junction of sulphuric acid and ferric chloride solution in
due to presence of deoxy ribose sugars in the sample. DNA is composed of deoxy-sugars
hence formation of blue-brown colour ring in test-tube indicates presence of DNA in it.

Experiment -4
Objective:
Quantitative estimation of ‘double stranded (Ds)-DNA’ by Ultra-violet-Spectroscopy
Or
Quantitative estimation of ‘double stranded (Ds)-DNA’ using Beer-Lambert Law.
Principle:
Using the Beer-Lambert Law it is possible to determine the concentration of given double
stranded DNA solution. Purines and pyrmidines nucleotide of DNA absorbs at ultra-violet
radiations of 260 nm. A solution of DNA obeys Beer-Lambert law, which says that
absorbance of given solution is directly proportional to its concentration and path length of
the solution.
At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is
0.020 μg-per ml-per cm.
For ds-DNA:
1 Absorbance unit (A260) or O.D. value for dsDNA = 50 µg
Concentration of ds-DNA can be calculated using following formula after taking the
absorbance at 260nm.
DNA concentration (μg/ml) = OD260 X dilution factor X 50 μg/ml

Requirement:
 1. Purified DNA.
2. Spectrophotometer
3. Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given DNA sample in 1 ml TE buffer.
2. Dilute this DNA sample to 1:20 by adding 100μl of DNA solution to 1900μl of TE
buffer.
3. Swithch -on spectrophotometer at least 20 minute before taking the O.D. of DNA
4. Set the spectrophotometer at 260 nm wavelength.
5. Put 2 ml TE buffer in quartz cuvette and take its O.D.
6. Set the O.D. of TE buffer to zero
7. Now took 2 ml of diluted DNA solution in quartz cuvette and note it O.D.

Observation:
Dilution factor of DNA solution= 1:20
O.D. of given ds-DNA sample at 260 nm= 0.75.

Result :
Using the formula given above for calculating the concentration of ds-DNA, concentration of
given DNA sample is:
DNA concentration (μg/ml) = OD260 X dilution factor X 50 μg/ml
= 0.75 X 20 X 50 μg/ml
= 75.0 μg/ml

Experiment -5
Objective:
Quantitative estimation of ‘single stranded (Ss)-DNA’ by Ultra-violet-Spectroscopy
Or
Quantitative estimation of ‘single stranded (Ss)-DNA’ using Beer-Lambert Law.
Principle:
Using the Beer-Lambert Law it is possible to determine the concentration of given single
stranded DNA solution. Purines and pyrmidines nucleotide of DNA absorbs at ultra-violet
radiations of 260 nm. A solution of DNA obeys Beer-Lambert law, which says that
absorbance of given solution is directly proportional to its concentration and path length of
the solution.
At a wavelength of 260 nm, the average extinction coefficient for single-stranded DNA is
0.027 μg-per ml-per cm.
For Ss-DNA:
1 Absorbance unit (A260) or O.D. value for SsDNA = 33 µg
Concentration of Ss-DNA can be calculated using following formula after taking the
absorbance at 260nm.
DNA concentration (μg/ml) = OD260 X dilution factor X 33 μg/ml

Requirement:
 1. Purified Ss DNA. For covering ds-DNA into Ss-DNA heat the DNA solution upto
80°C.
2. Spectrophotometer
3. Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given DNA sample in 1 ml TE buffer.
2. Dilute this DNA sample to 1:25 by adding 100μl of DNA solution to 2400μl of TE
buffer.
3. Switch-on spectrophotometer at least 20 minute before taking the O.D. of DNA
4. Set the spectrophotometer at 260 nm wavelength.
5. Put 2 ml TE buffer in quartz cuvette and take its O.D.
6. Set the O.D. of TE buffer to zero
7. Now took 2 ml of diluted DNA solution in quartz cuvette and note it O.D.

Observation:
Dilution factor of DNA solution= 1:25
O.D. of given Ss-DNA sample at 260 nm= 0.80

Result :
Using the formula given above for calculating the concentration of Ss-DNA, concentration of
given DNA sample is:
DNA concentration (μg/ml) = OD260 X dilution factor X 33 μg/ml
= 0.80 X 25 X 33 μg/ml
= 66.0 μg/ml

Experiment -6
Objective:
Quantitative estimation of ‘RNA’ by Ultra-violet-Spectroscopy
Or
Quantitative estimation of ‘RNA’ using Beer-Lambert Law.
Principle:
Using the Beer-Lambert Law it is possible to determine the concentration of given RNA
solution. Purines and pyrmidines nucleotide of RNA absorbs at ultra-violet radiations of 260
nm. A solution of RNA obeys Beer-Lambert law, which says that absorbance of given
solution is directly proportional to its concentration and path length of the solution.
At a wavelength of 260 nm, the average extinction coefficient for single-RNA is 0.025 μg-per
ml-per cm.
For RNA:
1 Absorbance unit (A260) or O.D. value for RNA = 40 µg
Concentration of Ss-DNA can be calculated using following formula after taking the
absorbance at 260nm.
RNA concentration (μg/ml) = OD260 X dilution factor X 40 μg/ml

Requirement:
1. Purified RNA.
2. Spectrophotometer
 3. RNase free Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given RNA sample in 1 ml of RNase free TE buffer.
2. Dilute this DNA sample to 1:10 by adding 100μl of RNA solution to 900μl of TE
buffer.
3. Switch-on spectrophotometer at least 20 minute before taking the O.D. of RNA
4. Set the spectrophotometer at 260 nm wavelength.
5. Put 2 ml TE buffer in quartz cuvette and take its O.D.
6. Set the O.D. of TE buffer to zero
7. Now took 2 ml of diluted RNA solution in quartz cuvette and note it O.D.

Observation:
Dilution factor of RNA solution= 1:10
O.D. of given RNA sample at 260 nm= 0.90

Result :
Using the formula given above for calculating the concentration of RNA, concentration of
given RNA sample is:
RNA concentration (μg/ml) = OD260 X dilution factor X 40 μg/ml
= 0.90 X 10 X 40 μg/ml
= 36.0 μg/ml

Experiment -7
Objective:
Determination of purity of ‘DNA sample’ by Ultra-violet-Spectroscopy and checking the
contamination of protein in it.
Principle:
Using the Beer-Lambert Law it is possible to determine the presence of contaminants like
proteins, phenol, trizol, carbohydrates, lipids, thiocynate and particulate matters in given
DNA solution. Purines and pyrmidines nucleotide of DNA absorbs at ultra-violet radiations
of 260 nm. While protein aminoacids absorbs specifically at 280nm. Hence a ratio of
A260/A280 can be used to determine the presence of protein contamination.
For a good quality purified DNA sample ratio of A260/A280 must be 1.8 or more. If value of
A260/A280 is less than 1.8, then it indicates contamination of protein in given DNA sample.

Requirement:
1. Two Purified DNA samples labelled sample-A and sample-B.
2. Spectrophotometer
3. Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given DNA sample in 2 ml of TE buffer.
2. Switch-on spectrophotometer at least 20 minute before taking the O.D. of DNA
3. Set the spectrophotometer at 260 nm wavelength.
 4. Put 2 ml TE buffer in quartz cuvette and take its O.D.
5. Set the O.D. of TE buffer to zero
6. Now took 2 ml of sample- A DNA solution in quartz cuvette and note it O.D. (at A260)
7. Wash the cuvette, put 2 ml of sample-B DNA solution and note its O.D. at same
wavelength (at A260)
8. Set the spectrophotometer at 280 nm wavelength.
9. Put 2 ml TE buffer in quartz cuvette and take its O.D.
10. Set the O.D. of TE buffer to zero
11. Now took 2 ml of sample- A DNA solution in quartz cuvette and note it O.D. (at A280)
12. Wash the cuvette, put 2 ml of sample-B DNA solution and note its O.D. at same
wavelength (at A280)

Observation:
• For sample A
O.D. of given DNA sample at 260 nm= 0.90
O.D. of given DNA sample at 280 nm= 0.50

• For sample-B
O.D. of given DNA sample at 260 nm= 0.70
O.D. of given DNA sample at 280 nm= 0.45

Result :
• For sample A
Ratio of A260/A280 = 0.90/0.50
= 1.8

• For sample B
Ratio of A260/A280 = 0.70/0.45
= 1.55

Conclusion:
For sample-A: ratio of A260/A280 is 1.8, which indicates that this DNA is pure and free form
protein contamination.
For sample-B: ratio of A260/A280 is 1.55, which indicates that this DNA is not pure and
protein contamination is present in it.

Experiment -8
Objective:
Determination of purity of ‘RNA sample’ by Ultra-violet-Spectroscopy and checking the
contamination of protein in it.
Principle:
Using the Beer-Lambert Law it is possible to determine the presence of contaminants like
proteins, phenol, trizol, carbohydrates, lipids, thiocynate and particulate matters in given
DNA solution. Purines and pyrmidines nucleotide of RNA absorbs at ultra-violet radiations
of 260 nm. While protein aminoacids absorbs specifically at 280nm. Hence a ratio of
A260/A280 can be used to determine the presence of protein contamination.
For a good quality purified RNA sample ratio of A260/A280 must be 2.0 or more. If value of
A260/A280 is less than 2.0, then it indicates contamination of protein in given RNA sample.

Requirement:
1. Two Purified RNA samples labelled sample-A and sample-B.
2. Spectrophotometer
3. RNase free Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given DNA sample in 2 ml of RNase free TE buffer.
2. Switch-on spectrophotometer at least 20 minute before taking the O.D. of RNA
3. Set the spectrophotometer at 260 nm wavelength.
4. Put 2 ml TE buffer in quartz cuvette and take its O.D.
5. Set the O.D. of TE buffer to zero
6. Now took 2 ml of sample- A RNA solution in quartz cuvette and note it O.D. (at A260)
7. Wash the cuvette, put 2 ml of sample-B RNA solution and note its O.D. at same
wavelength (at A260)
8. Set the spectrophotometer at 280 nm wavelength.
9. Put 2 ml TE buffer in quartz cuvette and take its O.D.
10. Set the O.D. of TE buffer to zero
11. Now took 2 ml of sample- A RNA solution in quartz cuvette and note it O.D. (at A280)
12. Wash the cuvette, put 2 ml of sample-B RNA solution and note its O.D. at same
wavelength (at A280)

Observation:
• For sample A
O.D. of given RNA sample at 260 nm= 0.85
O.D. of given RNA sample at 280 nm= 0.50

• For sample-B
O.D. of given RNA sample at 260 nm= 0.70
O.D. of given RNA sample at 280 nm= 0.35

Result :
• For sample A
Ratio of A260/A280 = 0.85/0.50
= 1.7

• For sample B
Ratio of A260/A280 = 0.70/0.35
= 2.0

Conclusion:
For sample-A: ratio of A260/A280 is 1.7, which indicates that this RNA sample is not pure and
protein contamination is present in it.
For sample-B: ratio of A260/A280 is 2.0, which indicates that this RNA sample is pure and free
form protein contamination.

Experiment -9
Objective:
Determination of purity of ‘DNA sample’ by Ultra-violet-Spectroscopy and checking the
contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol” in it.
Principle:
Using the Beer-Lambert Law it is possible to determine the presence of contaminants like
proteins, phenol, trizol, carbohydrates, lipids, thiocynate and particulate matters in given
DNA solution. Purines and pyrmidines nucleotide of DNA absorbs at ultra-violet radiations
of 260 nm. While Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol absorbs
specifically at 230nm. Hence a ratio of A260/A230 can be used to determine the presence of
protein contamination.
For a good quality purified DNA sample ratio of A260/A230 must be 2.0 or more. If value of
A260/A230 is less than 2.0, then it indicates contamination of “Phenol/
Thiocyanate/carbohydrate/organic compounds/Trizol" in given DNA sample.

Requirement:
1. Two Purified DNA samples labelled sample-A and sample-B.
2. Spectrophotometer
3. Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given DNA sample in 2 ml of TE buffer.
2. Switch-on spectrophotometer at least 20 minute before taking the O.D. of DNA
3. Set the spectrophotometer at 260 nm wavelength.
4. Put 2 ml TE buffer in quartz cuvette and take its O.D.
5. Set the O.D. of TE buffer to zero
6. Now took 2 ml of sample- A DNA solution in quartz cuvette and note it O.D. (at A260)
7. Wash the cuvette, put 2 ml of sample-B DNA solution and note its O.D. at same
wavelength (at A260)
8. Set the spectrophotometer at 230 nm wavelength.
9. Put 2 ml TE buffer in quartz cuvette and take its O.D.
10. Set the O.D. of TE buffer to zero
11. Now took 2 ml of sample- A DNA solution in quartz cuvette and note it O.D. (at A230)
12. Wash the cuvette, put 2 ml of sample-B DNA solution and note its O.D. at same
wavelength (at A230)

Observation:
• For sample A
O.D. of given DNA sample at 260 nm= 0.83
O.D. of given DNA sample at 230 nm= 0.50
 • For sample-B
O.D. of given DNA sample at 260 nm= 0.63
O.D. of given DNA sample at 230 nm= 0.31

Result :
• For sample A
Ratio of A260/A230 = 0.83/0.50
= 1.66

• For sample B
Ratio of A260/A230 = 0.63/0.31
= 2.03

Conclusion:
For sample-A: ratio of A260/A230 is 1.66, which indicates that this DNA sample is not pure
and contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol” is
present in it.
For sample-B: ratio of A260/A230 is 2.03, which indicates that this DNA sample is pure and
free form contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol”.

Experiment -10
Objective:
Determination of purity of ‘RNA sample’ by Ultra-violet-Spectroscopy and checking the
contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol” in it.
Principle:
Using the Beer-Lambert Law it is possible to determine the presence of contaminants like
proteins, phenol, trizol, carbohydrates, lipids, thiocynate and particulate matters in given
RNA solution. Purines and pyrmidines nucleotide of RNA absorbs at ultra-violet radiations
of 260 nm. While Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol absorbs
specifically at 230nm. Hence a ratio of A260/A230 can be used to determine the presence of
protein contamination.
For a good quality purified DNA sample ratio of A260/A230 must be 2.2 or more. If value of
A260/A230 is less than 2.2, then it indicates contamination of “Phenol/
Thiocyanate/carbohydrate/organic compounds/Trizol" in given DNA sample.

Requirement:
1. Two Purified RNA samples labelled sample-A and sample-B.
2. Spectrophotometer
3. RNase free Tris-EDTA (TE) buffer
4. Quartz cuvette
5. Micropipette

Procedure:
1. Dissolve given RNA sample in 2 ml of RNase free TE buffer.
2. Switch-on spectrophotometer at least 20 minute before taking the O.D. of RNA
3. Set the spectrophotometer at 260 nm wavelength.
4. Put 2 ml TE buffer in quartz cuvette and take its O.D.
 5. Set the O.D. of TE buffer to zero
6. Now took 2 ml of sample- A RNA solution in quartz cuvette and note it O.D. (at A260)
7. Wash the cuvette, put 2 ml of sample-B RNA solution and note its O.D. at same
wavelength (at A260)
8. Set the spectrophotometer at 230 nm wavelength.
9. Put 2 ml TE buffer in quartz cuvette and take its O.D.
10. Set the O.D. of TE buffer to zero
11. Now took 2 ml of sample- A RNA solution in quartz cuvette and note it O.D. (at A230)
12. Wash the cuvette, put 2 ml of sample-B RNA solution and note its O.D. at same
wavelength (at A230)

Observation:
• For sample A
O.D. of given RNA sample at 260 nm= 0.85
O.D. of given RNA sample at 230 nm= 0.37

• For sample-B
O.D. of given RNA sample at 260 nm= 0.63
O.D. of given RNA sample at 230 nm= 0.40

Result :
• For sample A
Ratio of A260/A230 = 0.85/0.37
= 2.29

• For sample B
Ratio of A260/A280 = 0.63/0.40
= 1.575

Conclusion:
For sample-A: ratio of A260/A230 is 2.29, which indicates that this RNA sample is pure and
free form contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol”.

For sample-B: ratio of A260/A230 is 1.575, which indicates that this RNA sample is not pure
and contamination of “ Phenol/ Thiocyanate/carbohydrate/organic compounds/Trizol” is
present in it.