Isolation, quantification of nucleic acids from wheat and synthesis of cDNA. Introduction List of Genotypes DNA Isolation (CTAB method) Qualitative check of DNA- Gel electrophoresis Quantitative test of DNA- Spectrophotometer Protocol for RNA Isolation RNA Confirmation Normalization of RNA cDNA Synthesis Protocol for DNA Isolation of plant 50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish. Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer. 1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube. The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins) The tubes were cooled for 10 minutes in ice. The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins. After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added. Then mixed gently for 5-7 mins by inverting the tubes. Again centrifuged for 10 mins @10,000 rpm After centrifugation, three layers were observed in the tube. a) aqueous phase i.e. DNA+RNA b) protein coagulate c) organic phase i.e. Chloroform Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight. Following day, tubes were again centrifuged @10,000rpm for 10 mins. The supernatant was discarded without disturbing the DNA pellet. 70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins. Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded. Pellet (DNA Precipitate) was air dried for 10 mins. Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C. 1g of analytical grade Agarose was weighed. 100 mL of autoclaved 1X TBE was added in flask. Now heated on the oven until the solution becomes transparent. Solution was allowed to cool down to 60℃. 2 μL of Ethidium Bromide (EtBr) is added in the flask. Melted agarose gel was poured into the casting tray along with comb. Any bubble in the gel was removed. After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank. Once gel got solidified, it was transferred it into gel tank. A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only. Then the mixture (sample +loading dye) was loaded into the well. Then electrophoretic unit was run at 90 volt for 50-55 mins. After that gel was put into the Gel Doc to see the DNA band (using UV light). Bright colour band were observed as in the figure. Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.

1. Dr. Rajendra Prasad Central Agricultural
University, Pusa
Project Advisor Presented By
Dr. Rajeev Kumar Asjad Raza
Associate Professor 1905101013
Isolation, quantification of nucleic
acids from wheat and synthesis of
cDNA
Department of Agricultural Biotechnology and Molecular Biology
College of Basic Sciences and Humanities.

2. This Presentation Includes:-
 Introduction
 List of Genotypes
 DNA Isolation (CTAB method)
 Qualitative check of DNA- Gel electrophoresis
 Quantitative test of DNA- Spectrophotometer
 Protocol for RNA Isolation
 RNA Confirmation
 Normalization of RNA
 cDNA Synthesis

3. Introduction
 Kingdom:- Plantae
 Family: – Poaceae
 Genus: – Triticum
 Species: – aestivum
 Botanical Name:-Triticum aestivum
 Common name:- Wheat
 Chromosome no :- 2n=42
 Ploidy level = 6x
 Genome size :- ~17 Gigabases

4. List of wheat genotypes
Sr. No. Genotype
1 DH5-239
2 DBW-187
3 Super-303
4 DH12-70
5 DH5-164
6 DH12-170
7 DBW-252
8 Dharward Dry

5. Begins…

6. DNA Isolation Using CTAB Method
Materials and chemicals required:
1)Plant sample (Leaf)
2)Centrifuge tubes
3)70% Ethanol
4)Extraction buffer-
a) CTAB
b) Tris-HCl
c) EDTA
d) NaCl
e) β- Mercaptoethanol
f) Polyvinlypyrolidone
g) Distilled water
5) Mortar –pestle
6) Chloroform- isoamylalcohol (24:1)
7) Chilled Isopropanol
8) Storage buffer (TE-1X)
Fig: Autoclaved reagents

7. Preparation of Extraction Buffer
Sl.No. Components Stock solution Working solution Amount taken
1 Tris HCL 1M 0.1M 3mL
2 EDTA 0.5M 25mM 1.5mL
3 NaCl 5M 1.5M 9mL
4 CTAB 10% 2% 0.6mL
5 PvP 10% 2% 0.6mL
6 Distilled water 15.3mL
 To prepare 30mL extraction buffer so after adding all the
components distilled water was added (30mL– 14.7mL =15.3mL)
 After preparation, it was put in warm water in water bath for 20-25
mins.
 Before use, 0.3ml β-mercaptoethanol was added to the working
solution.

8. :-
Protocol for DNA Isolation of plant
1. 50-100mg (2-3) young leaves were collected, then washed with tap water
followed by distilled water in petri dish.
2. Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by
taking 1mL extraction buffer.
Fig: Grinding of sample using mortar and pestle

9. Continued…
3. 1mL (1000μL) of extraction buffer was again added to collect paste
from mortar pestle & then transferred to the 2 mL micro centrifuge
tube.
4. The sample in the tube is incubated at 65°C in water bath for 35-45
mins. (Contents in the tube was mixed by inverting at an interval for
5-10 mins)
5. The tubes were cooled for 10 minutes in ice.
Fig: Inverting of tubes kept in water
bath

10. 6. The sample of equal vol (2mL) was centrifuged @14,000 rpm for
10 mins.
7. After that the supernatant was transferred to new 2 mL centrifuge
tube and equal volume (as of sample) of chloroform: Isoamyl
alcohol (24:1) was added.
8. Then mixed gently for 5-7 mins by inverting the tubes.
9. Again centrifuged for 10 mins @10,000 rpm
Continued…

11. 10.After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
11.Again the supernatant (aqueous phase) was collected in 1.5mL tube
and equal volume of ice-cold isopropanol was added and stored in -
20°C overnight.
12.Following day, tubes were again centrifuged @10,000rpm for 10
mins.
13.The supernatant was discarded without disturbing the DNA pellet.
Continued…

12. 14. 70% ethanol is taken and 0.5mL of it was added to the sample
and mixed by tapping for 5 mins.
15. Again centrifuged @10,000rpm for 10 mins and the supernatant
was discarded.
16. Pellet (DNA Precipitate) was air dried for 10 mins.
17. Then dissolved in 50μL TE-1X Buffer and the sample was stored
at -20°C.
Continued…
Fig: kept for Air dry

13. Qualitative check of DNA-
Gel electrophoresis
To check whether DNA is present in the sample or not, Gel electrophoresis
was carried out.
Gel electrophoresis-
Principle:-Gel electrophoresis separates DNA fragments by size in a
solid support medium (an agarose gel). The rate of migration is
proportional to size: smaller fragments move more quickly, and wind up at
the bottom of the gel. DNA is visualized by including in the gel an
intercalating dye, Ethidium Bromide.
Materials and chemicals required-
1) 1g Agarose
2) 100 ml of 1X TBE buffer
3) Ethidium bromide
4) Conical flask and oven
5) Casting tray and electrophoresis unit.
6) Running buffer (TBE-1X)-1L

14. Agarose gel (1%)
• 1g of analytical grade Agarose was weighed.
• 100 mL of autoclaved 1X TBE was added in flask.
• Now heated on the oven until the solution becomes transparent.
• Solution was allowed to cool down to 60℃.
• 2 μL of Ethidium Bromide (EtBr) is added in the flask.
Fig: 1% Agarose Gel

15. Procedure
• Melted agarose gel was poured into the casting tray along with
comb.
• Any bubble in the gel was removed.
• After solidification of gel, comb was removed gently and then
running buffer was added in the electrophoretic tank.
• Once gel got solidified, it was transferred it into gel tank.
• A parafilm was taken and on it 2μL loading dye and 3μL sample was
taken, gently mixed with the pipette tip only.
• Then the mixture (sample +loading dye) was loaded into the well.
Fig: Sample loading in well

16. • Then electrophoretic unit was run at 90 volt for 50-55 mins.
• After that gel was put into the Gel Doc to see the DNA band
(using UV light).
• Bright colour band were observed as in the figure.
Continued…
Fig: DNA band seen under Gel Documentation system
1 5 6 7 8
2 3 4

17. Nanodrop Spectrophotometer- To quantify the amount of DNA
sample. Nucleic acids absorb light at a wavelength of 260 nm. If
a 260 nm light source shines on a sample, the amount of light that
passes through the sample can be measured and the amount of
light absorbed by the sample can be inferred.
Steps to be followed:-
1) μCuvette was wiped with 70% ethanol then 1 μL of TE (1X) is
put onto that and the blank was set.
2) Then again wiped and sample was loaded and the absorbance
was recorded.
 A 260/280 ratio of ~1.8 is generally accepted as “pure”
for DNA;
 A ratio of ~2.0 is generally accepted as “pure” for RNA.
3)This step was carried out further for all the samples.
Spectrophotometric analysis of DNA

18. This shows the A260/A280 is 1.86
And amount of DNA present in the sample is 203.4 μg/mL
Continued…
Fig: Graph showing absorbance (Spectrophotometer reading)

19. RNA Isolation Protocol
1. Few (100-150mg) young leaves were ground into fine powder
using liquid Nitrogen.
2. 500 µL Tri-Extract solution was added into the fine power.
3. The grounded tissue was transferred to micro centrifuge tube.
4. 500 µL Tri-Extract was added into tube and mix well.
5. Incubated for 15 mins at room temperature.
Fig:- Leaves grinded under LN2

20. Continue…
6. 200 µL of chloroform was added for each 1mL of Tri-Extract to
the tube and mix well.
7. Incubated for 3 minutes.
8. Centrifuged at 12,000rpm for 15 min at 4°C.
9. Mixture got separated into a lower chloroform phase, a cloudy
white interphase, and a colorless upper aqueous phase.
10. Aqueous phase transferred to fresh micro centrifuge tube.
11. Added 0.5 ml of ice-cold isopropanol per 1mL of Tri-Extract
and gently mix.
12. Incubated overnight at -20°C.

21. Continue…
13. Centrifuged at 12,000rpm for 10 mins at 4°C.
14. Supernatant was discarded gently to get RNA pellets.
15. RNA pellet was washed by adding 500 µL of 70 % ethanol
and centrifuging at 7500 rpm for 5 minutes at 4°C.
16. Wash was discarded and the RNA pellets were air dried at
room temperature on ice.
17. 40 µL nuclease free water was added to the air dried pellets
to dissolve the pellet and solution was stored at -80°C until
further use.

22. RNA Confirmation
• 2µL of RNA was taken from each sample and 4µL of
loading dye was added and mixed well
• The sample was loaded into 1% Agarose gel for 45 minutes.
• DNA was visualized in gel documentation unit under UV
light.
• Isolated RNA quality and quantity was checked using
Nanodrop.
Fig:- RNA under gel doc
28S
18S
L. Ladder(1Kb)
1. DH5-239
2. DBW-187
L 1 2

23. cDNA Synthesis
Content for RT reaction
mixture
Amount
2x RT Easy Mix 5µL
Oligo(dT) Primer 0.5µL
Template (RNA) X µL (2ug of RNA)
RNase-Free ddH2O (4.5-X) µL
Total volume 10µL
1. Preparation of RT reaction mixture for cDNA synthesis.
2. Reaction mixture was incubated at 42°C for 20 min for
reverse transcription then at 85°C for 5 min for enzyme
inactivation.

24. cDNA confirmation with actin
L- 100 bp ladder, GBiosciences
1. DH5-239
2. DBW-187
L 1 2
136bp