SDMVM’S COLLEGE OF

AGRICULTURAL BIOTECHNOLOGY.
Course No. :READY-471
Course Title: In House Skill Development Module

Departments
Plant Biotechnology
Bioinformatics
Microbial and Environmental Biotechnology

Speaker
Student Name : WAGH SHUBHAM SHRIRAM.
Reg. No. : 2017BTGT070
 MOLECULAR BIOLOGY

Practical -1: DNA Extraction

Aim : To study the isolation of plant genomic DNA by using modified CTAB method
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 syst
Ethidium Bromide solution
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
(EthylenediaminetetrAcetic acid Di-sodium salt)
28.0 ml 5 M NaCl 40.0 ml H2O 1 g PVP 40
(polyvinyl pyrrolidone (vinylpyrrolidine
homopolymer) Mw 40,000) Adjust all to pH 5.0
with HCL and make up to 100 ml with H2
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.
Method :
Plant samples can be prepared by cryogenically grinding
tissue in a mortar and pestle after chilling in liquid nitrogen.
Freeze dried plants can be ground at room temperature. In
either case, a fine powder is best for extracting DNA.
1.Transfer the ground plant tissue to a polypropylene tube.
2.For every 100 mg of homogenized tissue add 500 µl of
CTAB Buffer. Mix and thoroughly vortex.
3.Place the tube in a 60°C water bath for 30 minutes.
4.Centrifuge the homogenate for 5 minutes at 14,000 x g.
5.Transfer supernatant to a new tube.
6.Add 5 µl of RNase A solution and incubate at 37°C for 20
minutes.
7.Add an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1).
8.Vortex for 5 seconds then centrifuge the sample Labwares required
for 1 minute at 14,000 x g to separate the phases.
9.Transfer the aqueous upper phase to a new tube. 1. Scissors,
Repeat this extraction until the upper phase is clear. 2. Disposable gloves,
10.Transfer the upper aqueous phase to a new tube. 3. Paper towels/Kimwipes,
11.Add 0.7 volume cold isopropanol and incubate at
-20°C for 15 minutes to precipitate the DNA. 4. Ice bucket,
12.Centrifuge the sample at 14,000 x g for 10 5. Micro-centrifuge tubes,
minutes. 6. Micro pipettes and
13.Decant the supernatant without disturbing the
7. Micro pipettes tips
pellet and subsequently wash with 500 µl ice cold
70% ethanol. (1000 µl, 2- 20 µl, 0.5 - 2 µl)
14.Decant the ethanol. Remove the residual ethanol 8. Labeling/Colored tapes
by drying in a SpeedVac. 9. Reagent bottles (500 ml/200ml)
15.Dry the pellet long enough to remove alcohol, but
without completely drying the DNA. 10. Marking pens
16.Dissolve the DNA pellet in 20 µl TE buffer (10 11. Beakers
mM Tris, pH 8, 1 mM EDTA). The pellet may need 12. Eppendorf racks
to be warmed, in order to dissolve.
13. Tissue paper rolls

1. Thread like DNA start appearing with addition of pre-chilled

ethanol/iso-propanol in respective stage.
2. DNA pellet of transparent to white colour is obtained after
final centrifugation
 MOLECULAR BIOLOGY

Practical-2
Qualitative estimation of genomic DNA
Aim : To study the qualitative estimation of DNA with the help of agarose gel electrophoresis.

Basic Principle
 It is critical that plant DNA must be of high molecular weight for molecular analysis (> 30 kb).
 The isolated DNA needs to be estimated for quality before starting of molecular biology experiments.
 To check this you will need to run an aliquot (2l) of each sample on 0.8 % agarose gel.
 High molecular weight DNA will reveal single band near high molecular weight size band of molecular weight
standard.
 If a second band is revealed at the lower portion of the gel, it indicates contamination of RNA and a smear in the gel
shows protein contamination and degraded DNA.
Chemicals required Material : Genomic DNA
• Tris (1M) Labwares required
• EDTA (0.5M) Agarose gel electrophoresis unit with power pack,
• Glacial Acetic Acid Conical flask,
Micropippetes and tips.
• Autoclaved
distilled water
(ADW)
• Standard DNA
• Ethidium Bromide
• Ethanol (70%)
• Agarose
• DNA loading dye Apparatus required
(6X – orange Gel Documentation system
loading dye)
• TAE (1X) buffer
Preparation of Reagents Preparation of 0.8 % agarose gel
10X TAE 1. Take 100 ml 1X TAE buffer in a conical flask

TAE Buffer – pH 8.2 1000 2. Add 0.8 g agarose to 100 ml 1X TAE and allow agarose
m to melt by heating the solution (microwave oven/ heating
mantle)
Tris Base 242 g
3. Gently stir the solution intermittently till a transparent
Glacial acetic acid 57.1 ml
gel like mixture is obtained
0.5M EDTA (pH 8.0) 100 ml
* Finally makeup volume to 1000ml 4. Do not allow to overheat and bubble formation

1X TAE 5. Allow to cool to warm and add 3 µl Ethidium Bromide

with the help of a pipette (Stock concentration - 10
Prepare 1X TAE as working mg/ml; Working concentration - 1 μg/ml.)
solution from stock of 10X
TAE by taking 10 ml of 10X
TAE and 90 ml double
distilled water (DDW)
6. Pour gel on gel casting tray (with suitable comb and sealed) 11. Run the gel by attaching cod to
7. Allow to solidify then gently remove the comb and seals. the power pack for 20 min
8. Place the gel on electrophoresis unit ‘tank’ along with casting tray. 12. Gel documentation by using gel
doc system
9. Mix loading dye with DNA as (5 µl dye + 2 µl DNA).
10. Load DNA samples (mixed with loading lye) over the well formed
by comb

*EtBr: Highly carcinogenic/ mutagenic – any contact with skin, eyes or

direct inhalation must be avoided.
Results:
During agarose gel electrophoresis of genomic
DNA, it was found that the resulting DNA
fragments are visible as clearly defined bands.
But some RNA contamination was found in
sample 5 and 8 whereas, the sample 2 and 3
showing the protein and degraded DNA.
The DNA standard or ladder separated to a
degree that allows for the useful determination of
the sizes of sample bands.
 MOLECULAR BIOLOGY
Practical-3
PCR based RAPD analysis
Aim: To study the PCR amplification of DNA with particular RAPD primers at recommended annealing temperature
Basic Principle
1. Polymerase Chain reaction (PCR) is an in vitro method of nucleic acid synthesis by which a particular segment of
DNA can be specifically amplified.
2. PCR technology had a significant impact in almost all areas of molecular biology and modification of the basic
procedures has allowed developing numerous assays for detecting variation at the nucleotide level.
3. Random amplified polymorphic DNA, approach had been widely used in plant molecular biology. William et al.
(1990) have developed RAPD to detect genetic polymorphism in crop plants.
4. RAPD PCR is typically carried out using random oligonucleotide (decamers) primers (10 nt length) that flank the DNA fragment to be
amplified.
5. These primers anneal to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the
region between the primers.
6. Amplification is achieved through repeated cycles of denaturation, annealing and extension of the annealed primers with thermostable DNA
polymerase (Taq Polymerase).
 Chemicals required: Labwares required:
1. RAPD random primers (10 nt) 1. PCR tubes
2. dNTPs 2. Micro pipette
3. Template DNA, 3. Tips
4. PCR buffer 4. Mini cooler tray
5. Taq DNA polymerase, 5. Ice bucket
6. Agarose gel eletrophoresis reagents (1X TAE, Apparatus required
Ethidium Bromide, Gel loadng dye etc.)
PCR thermocycler Agarose gel
7. Ultra pure water.
electrophoresis unit with power pack

Material required
Sample DNA to be analysed (20 – 40 ng/µl)
Procedure
1. Label 0.2 ml PCR tubes for each genomic DNA and arrange the
tubes in the rack.

2. Add 2l of template DNA to the tubes already labeled and keep
in the PCR tube rack.

3. Prepare the following reaction cocktail in a 1.5 ml Eppendorf

tube for required number of reactions plus two reactions to
compensate the pipetting loss.
Components Concentration Quantity 7. Perform the PCR reaction in a thermocycler following the
programme given below:
Sterile H2O -  11.86 l
PCR buffer with MgCl2 10X 2.00 l
Ste Temperatu Duration Cycles Activity
dNTP mix 10mM 1.60 l
Primers 10mM 1.50 l ps re (0C) (min)
Taq Polymerase 1 U/l 0.80 l 1 95 0C 10:00 min Denaturation
1
Template DNA 20 - 50ng /l 2.00 l 2 95 0C 00:15 sec Denaturation
Total  - 20.00l 3 52 0C 00:30sec  36 Annealing
4. N.B. While preparing the PCR reactions, it is important to keep the 4 72 0C 1:30 min Extension
reactions in ice, add the components in the order as indicated.
5 72 0C 10:00 min Final
1
5. Add 18l of the reaction mixture to the PCR tube, which is already extension
loaded with 2l of template DNA making the final volume to 20l
6 4 0C Till further Storage
6. Mix by flicking the tube repeatedly with your finger and then
1
use
centrifuge for a few seconds to bring the contents to the bottom of 8. Load the tubes in PCR thermcycler machine and set PCR as following:
the tube (do not use the vortex mixer to mix the enzyme mixture as
this inactivate the enzyme).
10. Take out samples after completion of
cycles and resolve in agarose gel.
A. Load the RAPD products (10l) of individual reactions for
electrophoresis in 1.5% agarose gel prepared with 1X TAE buffer
(having ethidium bromide)
B. Run in 1X TAE buffer at 80 volt for 1 hours. Standard DNA
molecular weight marker must be added to one of the wells in the gel.

Lane 1: 100 bp DNA Ladder

Lane 2: RAPD Pattern of wheat-1 genomic
After completion of the PCR, perform agarose gel DNA
Observations: electrophoresis and compare the RAPD patterns of Lane 3: RAPD Pattern of wheat-2 genomic
the four different samples genomic DNA DNA Lane 4: RAPD Pattern of cotton-1
genomic DNA Lane 5: RAPD Pattern of cotton-
Results 2 genomic DNA
1.The RAPD pattern i.e. number and size of the amplified PCR product varies among
different DNA samples taken for study.
2.This happens due to the variation in their genomic DNA sequences and annealing sites
of the random primer.
3.By doing this experiment one can study the RAPD fingerprints for differentiating
individuals at genus and species level.
4.The RAPD patterns of wheat and cotton show significant differences but the RAPD
patterns within the species of same genus i.e. wheat-1, wheat-2 show less differences.
 PLANT TISSUE CULTURE
Practical-1: Preparation of MS nutrient medium

Aim : To study how to make the best type of MS media for micropropagation of different plants
Principle:
The basic nutritional requirements of cultured plant cells as well as plants are very similar. However, the nutritional composition varies
according to the cells, tissues, organs and protoplasts and also with respect to particular plant species. The appropriate composition of the
medium largely determines the success of the culture. A wide variety of salt mixtures have been reported in various media.
A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, growth regulators and other organic supplements. When
referring to a particular medium, the intention is to identify only the salt composition unless otherwise specified. Any number and concentration
of amino acids, vitamins, growth regulators and organic supplements can be added in an infinite variety of compositions to a given salt
composition in order to achieve the desired results.
Materials required: Glassware's, chemicals, pH meter, distilled water, autoclave.

Procedure
1. For MS media preparation autoclaved bottle of 1 litter capacity along with distilled water (800ml) was taken.
2. Then weight the 4.4 g media (HiMedia Laboratories Pvt. Ltd.) and dissolved in distilled water.
3. Weight maltose (1.5%) /sucrose (3%) to dissolve in distilled water.
4. Then magnetic stirred for uniform dissolving solution, after completion of the dissolving process pH maintained at 5.8.
(1N NaOH used for increase the pH of solution and 1N HCL used for decrease the pH of solution
5. After adjustment pH, make up the volume of 1 litter.
6. Then weight the agar-agar 8 gm add in the solution.
7. Prepared media was autoclaved at 15 psi for 20 minutes.
8. Then kept it in growth chamber for cooling the media.
9. Before pouring the media in plates add the hormones NAA, Kinetin, BAP (0.5mg/l: 0.5mg/l: 1.5mg/l) as required amount according to
treatment.
10. Then pouring the media in the laminar air flow in controlled condition.
Precautions:
Regular stirring is to be done while dissolving the agar.
Media should be dissolved in lower volume of around 800 ml and then volume should be made up to 1000ml.
Ensure appropriate pH of the medium before addition of gelling agent as acidic pH will lead to decreased gelation resulting in semi solid
flowing gel while alkaline pH will lead to formation of hardened gel.
Use of Distilled water / Tissue culture grade water is recommended for media preparation as tap water or lower grade water may lead to salt
precipitation and improper gelation.
Avoid preparation of concentrated solutions, as it will lead to precipitation of salts.

Storage and Shelf Life: Must be stored at 2-8°C in

air tight containers.
 PLANT TISSUE CULTURE

Practical-2
Aseptic manipulation of various explants
Aim : To study the sterilization process of explants used in micropropagation technique by using various chemicals.

SURFACE STERILIZATION OF EXPLANTS

The first important condition for the successful tissue culture procedures is the maintenance of aseptic
condition. Sterilization eliminates microorganism and thus avoids contamination by bacteria and fungi. To maintain an aseptic environment, all
culture vessels, media and instruments used in handling tissue, as well as the explant itself is should be surface sterilized. Plant material can be
surface sterilized by variety of chemicals. Some commonly used chemicals sterilants are as follows:
1 % sodium hypochlorite (NaOCl) :
It is generally available with 5 % active chlorine content, so 20 % can be used for normal sterilization.

Calcium hypochlorite Ca(OCl)2:

This comes in the powder form. Generally 100 ml of Ca(ClO)2 is used. The desired weight of hypochlorite is added in to the water, agitated for 10 min, allowed to
settle and the clarified filtered supernatant solution is used for sterilization. The filtrate is used immediately because of deliquescent (take up water) nature. Calcium
hypochlorite enters the plant tissue slowly as compared to sodium hypochlorite. The standard concentration used is of the order of 4 to 10 % and the soaking time
varies from 5 to 30 min.
Bromine Water:
1to 2% bromine water solution is used for the sterilization purpose.

Mercuric chloride:
It is dissolved in water to create the solution. Concentration of 0.01 to 0.1 % for 2 to 10 min, depending upon the tissue, is used. Mercuric
chloride is an extremely toxic substance for plant, so rinsing must be very thorough at least five times.
Alcohol: PROCEDURE
70 % alcohol is used for sterilization of plant material by dipping them
for a period of 30 sec to 2 min. Generally alcohol alone is not sufficient 1.Wash explant with tap water to remove soil and dust particles deposited on
surface.
to kill all the microorganisms and the plant material after alcohol
treatment is treated another chemical sterilant. 2.Transfer the washed explant into a glass beaker containing tap water; add few
drops of liquid detergent – Tween 20.
Antibiotic:
Cefotaxime antibiotic at 50 mg/L concentration in the nutrient 3.Cover beaker mouth with muslin cloth with the rubber band and keep under
running tap water for 1 hour to remove any waxy/ oily deposition on explant
medium is generally used to control bacterial infection. surface.
Explants after treatment with sterilants must be thoroughly
rinsed with sterile distilled because retention of such toxic 4.Wash it twice with distilled water.
chemicals will seriously affect the establishment of culture 5.Transfer the explant into laminar airflow hood for farther work to avoid
contamination.
REQUIREMENTS
6.Wash the above explant with sterile distilled water for thrice each washing
Reagents & Chemicals: should be for 3-4 minutes.
7. Treat it with 0.1% HgCl2 solution for 60 sec.
Tween 20 (liquid detergent) , 0.1% HgCl2 , 70% alcohol, sterile distilled
8. After treating it with disinfectant, wash it with sterile distil water for thrice,
water each washing should be for 3-4 minutes.
Glasswares 9. Wash with 70% alcohol for 30 seconds to remove water from the surface of
the explant.
Beakers, sterile petri plates, sterile blades, sterile forceps, muslin cloth
10. Transfer the sterile explant to a sterile petri-plate.

11. Cut the explant into small pieces of about 1 cm with sterile blade.

12. Now the explant is ready for inoculation.

Equipment
Laminar airflow hood, Autoclave
 PLANT TISSUE CULTURE

Practical-3
Micropropagation of important crops
Aim : To study the protocol for micropropagation technique
Principle:
Plant cells and tissues are totipotent in nature i.e., every individual plant cell or tissue has the same genetic makeup and capable of developing
along a "programmed" pathway leading to the formation of an entire plant that is identical to the plant from which it was derived.
The totipotency of the plant cells and tissues form the basis for in vitro cloning i.e., generation or multiplication of genetically identical plants
in in vitro culture.
The ability to propagate new plants from a cells or tissues of parent plant has many interesting possibilities.

 Micropropagation is used commercially to asexually propagate plants. Using micropropagation, millions of new plants can be derived from a
single plant.
 This rapid multiplication allows breeders and growers to introduce new cultivars much earlier than they could by using conventional
propagation techniques, such as cuttings.
 Micropropagation also can be used to establish and maintain virus-free plant stock. This is done by culturing the plant's apical meristem,
which typically is not virus-infected, even though the remainder of the plant may be.
 Once new plants are developed from the apical meristem, they can be maintained and sold as virus-free plant

Tissue culture is particularly useful for multiplication of
plants which are
1.Slow growing (turmeric, ginger, cardamom);
2.Cross- pollinated (coconut, teak, eucaluptus, cashew, mango and those which
show wide variation in the progeny);
3.Male-sterile lines (cotton, sorghum, pearlmillet);
4.Newly produced varieties (normally vegetatively propagated);
5.For multiplication of virus free plants by meristem cultures (sugarcane,
potatoes, tapioca, etc.).
Tissue culture is now being commonly used for clonal propagation of a large
number of horticultural plants.
The success of clonal multiplication in higher plants depends generally on 3 photoperiod and light intensity grows to form large number of
main stages:
STAGE 1: ESTABLISHMENT OF AN ASEPTIC CULTURE
STAGE 2: MULTIPLICATION
STAGE 3: ROOTING AND HARDENING OF PLANTS
STAGE 1: ESTABLISHMENT OF AN ASEPTIC
CULTURE
1.The explants taken from the plant has first to be made free of
microorganisms which would outgrow the plant tissue when
placed on a nutrient medium. This would result in the death of
the explants.
2.These surface contaminants, e.g. bacteria, fungi and yeast are
removed by surface sterilization prior to culture, but without
killing the plant tissue.
STAGE 2: MULTIPLICATION
1.The surface sterilized material when inoculated on sterile
nutrient media and incubated at 25±2ºC with a definite
shoots.
STAGE 3: ROOTING AND HARDENING OF PLANTS
1.The shoots obtained are carefully excised and transferred to a rooting
medium, preferably a liquid medium, containing an auxin and supported
on a filter paper platform in order to obtain rooting in these shoots.
2.These plants which have rooted and have developed secondary roots
with root hairs can be transferred to pots containing soil:vermiculite
mixture (1:1).
3.This mixture is pre-autoclaved for 1 hour at 15 psi and steamed for 3
days successively and cooled. The potted plants can be transferred to the
field where the first new leaf emerges.
 MULTIPLICATION BY SUBCULTURE AT STAGE 2 1. Initiation stage: A piece of plant tissue (called an explant) is
(a) cut from the plant, (b) disinfested (removal of surface
1. However, excised shoot tips can be inoculated on the same medium
contaminants), and (c) placed on a medium. A medium
used in stage 2 instead of the rooting media. By regular repetition of
typically contains mineral salts, sucrose, and a solidifying
this subculture procedure, high rates of multiplication can be achieved.
agent such as agar. The objective of this stage is to achieve
2. Vegetative multiplication of plants depends on various factors as an aseptic culture. An aseptic culture is one without
nutrient medium, agar concentration, photoperiod and light intensity, contaminating bacteria or fungi.
hydrogen ion concentration, size and source of the explants.
2. Multiplication stage: A growing explant can be induced to
Requirements: produce vegetative shoots by including a cytokinin in the
a) Equipments medium. A cytokinin is a plant growth regulator that
promotes shoot formation from growing plant cells.
 Conical flasks (100ml capacity)
3. Rooting or preplant stage: Growing shoots can be induced
 Test tubes (25mm*150mm) to produce adventitious roots by including an auxin in the
medium. Auxins are plant growth regulators that promote
 Petridishes (80mm diameter) root formation. For easily rooted plants, an auxin is usually
 Pair of forceps and scalpel (15 cm long) not necessary and many commercial labs will skip this step.

 Environmental growth cabinets adjusted to 25º±2ºC with 18hr 4. Acclimatization: A growing, rooted shoot can be removed
photoperiod and 1500lux intensity and 15º ± 2º and 600 lux light intensity. from tissue culture and placed in soil. When this is done, the
humidity must be gradually reduced over time because
 Shaker with 120rpm and 1000 lux light intensity. tissue-cultured plants are extremely susceptible to wilting
b) Culture media, washing solutions, sterilizing agents, Glass distilled water,
sterile glass distilled water, 0.5% HgCl2 solution and Detergent Medium
c) Source tissue
Procedure :-
a. Sterilization of glassware
b. Preparation and sterilization of media
c. Explants collection:
1. Select a twig (60-90 cm long, 10-15mm wide) from mature elite trees and cut, making sure that the twig contains many young axillary buds.
The length is important in selecting twig that do not wither before being brought to the laboratory.
2. Bring the twigs containing axillary bud to the laboratory, remove the leaves and cut them into small pieces of about 5-8 cm.
3. Transfer the buds to a sterile 250ml conical flask and surface sterilize the explants.
d. Culture of buds:
1. Keep sterile petri-dishes, scalpel, forceps and medium inside a sterile cabinet along with the flask containing surface-sterilized explants.
2. Transfer these explants into sterile petri dishes with the help of a pair of sterile forceps and cut these explants into small pieces of 10-15
mm each containing atleast one axillary bud.
3. Inoculate 2 pieces to each tube containing medium.
4. Incubate the tubes in an environment growth cabinet at 15º±2º and 500 lux light intensity for 72 hours.
5. Transfer the cultures after 72hr to another incubator maintained at 25º±2ºC with 16hr photoperiod and 1500lux intensity.
6. After 25 days, the young buds start sprouting.
7. When the sprouts are 10-15mm long, transfer them to liquid medium.
8. Incubate the flasks on a rotatory shaker at 120 rpm and 500 lux light intensity.
9. Observe the formation of multiple shoots after 10-15 days.
e. Multiplication by subculture:
1.Transfer the multiple shoots from the flask to a sterile
petridish aseptically.
2.Incubate the cultures in an environmental growth cabinet at
25º±2ºC and at 1000 lux light intensity (12 hr photo periods)
and observe the cultures regularly.
3.Observe the explants produces multiple shoots within 15
days.
4.Separate these shoots again aseptically and transfer to
medium used for root formation.
f. Transfer of plants to pots:
1.Remove the rooted plantlet from the tube and wash the
roots gently with tap water to remove any traces of medium.
2.Transfer the plantlets to soil: vermiculite (1:1) sterile
mixture in a pot.
3.Irrigate with about 20 ml of tap water.
4.Keep the pots in a growth cabinet at 25º±2ºC and at 1000
lux light intensity and water them.
5.Transfer the plants to the field after 8 days of hardening in
which 70-80% plants survive.

Results: Healthy and disease-free plants were obtained after

hardening under greenhouse conditions
 BIOINFORMATICS
Practical No. 1- Retrieval of nucleotide sequences from GenBank
 Aim: To retrieve a nucleotide sequence of interest from Genbank entry with Specific accession number.
Procedure:

1. Open the web browser and type the database address http://www.ncbi.nlm. nih.gov/genbank/ in the address bar.

2. Select nucleotide database from the All Databases

 3. Type the query (i.e., the accession number for which the nucleotide sequence has to be retrieved) in the search bar and click on search option (AF465255- Oryza sativa
cultivar Nipponbare gibberellin-20 oxidase gene)

4. Select the particular sequence which is required.

5. Select FASTA format in the display settings and click apply.

6. Copy the sequence data and paste it in the notepad.

 7. Save the notepad file in FASTA format.

8. Report the result: 6590 bp linear DNA of Oryza sativa cultivar Nipponbare gibberellin-20 oxidase gene submitted (02-JAN-2002) Cytogenetics,
National Institute of Agricultural Science and Technology, 249, Seodun-dong, Suweon, 441-707, Korea.
Result: Thus the nucleotide sequence (Accession Number: E01306.1) was retrieved from GenBank Database.
 BIOINFORMATICS
Practical No. 2 - BLAST ANALYSIS
AIM:- To identify the presence of given marker on particular BAC/PAC clones of chromosome.
Procedure - 3. Select the marker sequence for blast, directly blast this sequence or copy this
1. Open the GRAMENE home page, different type of tools are sequence and paste in blast portal.
on the home page of gramene, we select the marker tool.

Copy this sequence

for blast or directly
blast this sequence

2. Search the marker sequence for blast analysis. 4. Fill the necessary information in the blast portal and click on configure.
5. Click over configure and fill up the E-value which require less than zero as
shown below. click over Run option. 7. Alignment summary of the query sequence.

8, Click the hyperlink “A” to get this review alignment.

6.We get exact location of that given sequence or marker on chromosome
which is detected by rectangle as shown in figure
 9.Click over contig, it give accession no of that BAC clone, as shown in diagram .

10. By selecting the accession no. we get exact location of that BAC clone over chromosome where its sequence situated.

RESULT:-
The exact location of the given marker on particular BAC/PAC clone was identified by blast analysis.
 Practical No. 3 - NTedit and NTSYSpc

AIM- To analyse qualitative data and generate dandogram using NTedit and NTSYSpc programme.

Procedure-

1.Generate binary data by observing gel in excel sheet and save it in 97-2003 mocrosoft excel workbook.

2. Click over NTedit programm and open file in grid.

3. Saved data automatically import in the NTedit sheet then save it in particular location by giving extension NTS.

4. Open the NTSYSpc software and select the dissimilarity and then quantitative data.
5. Browse and select the file in the input file dilog box and give the choice of location in the output file dilog box then click on compute.

6. See the report.

 7. Open the new tab of software and click on cluster and then click on SHAN.

8. Copy previously save file in the inpute option and paste output file by giving new extension to it and compute it.
9. See the report.

10. Click on the left corner of the software (on dendogram logo).
 11. Set the tree plot option.

12. Save the dandogram metafile .

RESULT:-
Qualitative data can be differentiated on the basis of similarity/dissimilarity in the form of dendogram that can show
different clusters variation at different percent by using NTedit and NTSYSpc programme.
 MOCROBIAL AND ENVIRONMENTAL BIOTECHNOLOGY.
READY-471
(7TH ) NEW (BEVAREGE)

Theory:-
Peanut (Araches hypogeo) is important oilseed crop originated from South Africa,while India represented one of its leading product
nearly 14% & wooled peanut production. Now a days some of the life style changes & medical issues like cow’s milk allergy, lactose-
intolarance & hypercholestoria people were shifted into plant based non diary beverages. Peanut milk was developed from two
different peanut varieties viz TNAUCO6 & local variety. Proximate composition of local and TNAUCO6 variety of peanut was analysed &
the carbohydrate proper fat was high in TNAUCO6 peanut variety & its value was 26.7 (g/100g), 2789/100g, 38(g/100g) when
compared to local variety its value was 25.2 (g/100g) , 24.7 (g/100g), 39 (g/100g) respectively. Peamilk was extracted by 5 different
processing method . Fresh soaking,blanching,roasting & germination methods in both local & TNAUCO6 variety. Peanut milk prepared
without any treatment & processing was considered as a control peanut milk. Among the different treatment the best treatment was
selected base on sensory scores in each processing methods & both peanut variety.
Observation:-
fresh off white coloured peanut nutrabeverage prepared and different nutrient content were tested biochemically as
per fssai i.e. food safety & standard authority of India 2015 such as Reducing sugar , total protein & milk fats &
microbial analysis was also carried out e.g. Determination of total microbial plate count.
Result :-
Peanut milk beverage was prepared (photo)
 MOCROBIAL AND ENVIRONMENTAL
BIOTECHNOLOGY.
READY-471
7th NEW Module
Dept:- Microbial & Environment Biotech.
2) Formulation & production of Soy milk a nutra beverage
Requirement:-
Soyabean seed , mixer grinder , sieve or mustin cloth , distilled water.
Theory :-
Antioxident Defence
A study reported that fermented soymilk possesses high-cytotoxic activity towards cancer cells & antioxidant activity due to
higher aplyone form . Peptides derived from soy glycinit protein are one of the most abundant storage protein in
soybean.They have show a high degree of antioxidant activity soymilk is a very popular beverage with the Chinese,
Japanese& Western population. However its production has increased in USA due to lipoxygenase soyabeans and improved
processing methods that remove beany flavor of the milk . The health benefit of soyprotein to reduce cholesterol (heart
disease) has also caused an increased in soy milk sales as the USFDA now allow a health claim if the food contain at least
6.25g soyprotein with low levels of cholesterol , saturated fat & total fat . Soy milk is very important to Tofu producers
because it is the intermediate product in the manufacture of Tofu.
Requirement:-
Large seeds of soyabeans 200gms vanilla essence,autoclave, mustil cloth, petridishes , inculsator
Procuder :-
Soymilk is traditionally made by soaking soyabeans in water overnight then grinded the beans with water added during grinding full fat
flakes , grints or flour can be used to produce the soymilk slurry . The resulting slurry is boiled and stirred for 1-30 mins. This heating step
improves the ‘nutritional values’ of the milk and improves flavour ; by inactivating lipoxygenase & volatizing same of the off flovour
compounds that result during grinding heating also increase the shelf life of the milk by reducing microbial load . The heated slurry is then
filtered through a cloth or a nylon bag to separate the undespersible fiber reduce form the soyamilk . The resulting soy milk was flavoured
with vanilla essence and pasturized 63’c – 30 min or 145’f before packing aseptically.
Observation :-
Pale yellow colored , tasty and flavoured soymilk was prepared and biochemical tests were carried out such as reducing sugar , total
protein, milk fats and microbial analysis was also carried out e.g. determination of total microbial plate.
Result :-
Vanilla flavoured soymilk was prepared.
 EXPERIMENT NO. 3
POUR PLATE TECHNIQUE
Aim:
To isolate microorganism from soil using pour plate technique.

Principle:
The pour plate technique requires a serial dilution of the mixed culture. The diluted inoculum is then added to sterile molten agar tubes that have
been cooled to 42 to 45°C. The bacteria and agar medium are mixed well, and mixture is immediately poured into sterile Petri plate under aseptic
condition. The agar is allowed to solidify, trapping the bacteria at separate discrete position within matrix of medium. A series of agar plates
showing decreasing number of colonies resulting from the dilution procedure in the pour plate technique. Some of the organisms are trapped
beneath the surface of medium when it gels, and therefore both surface and subsurface colonies develop. In this technique, proper distribution
of colonies is observed; therefore, counting of microorganisms is possible.

Requirements: 4.composition: - For 1000ml

1. Soil dilutions. Peptone - 5gm
2. Sterile Petri plates. NaCl - 3gm
3. Nutrient agar Beef extract - 3gm
Distilled water- 1000ml
Agar - 20gm
PH - 7.0
Autoclave the media at 121°C, 151 pressure for 20 minutes.
 5. Sterile 9 ml distilled water in tubes.
6. Bunsen burner.
7. Incubator.
8. Sterile micropipette and sterile tips.
9. Ethanol.
Procedure:
1. Soil suspension was prepared in sterile distilled water.
2. Serial dilutions were made from 10¹ to 106
3. 0.1 ml of soil suspension was added in sterile Petri plates.
4. Then sterile molten nutrient agar (42° C to 45° C temperatures) was poured into
the plate & thoroughly mixed with the inoculum.
5. It was allowed to solidify and incubated in inverted positions at room temperature
for 24 hours.
6. Next day the colony characters were studied.
Result:
 
Surface as well as submerged colonies were observed.

Conclusion:
Using pour plate technique the microorganisms can be isolated.
 Educational Tour Report
SDMVMS College of Agril. Biotechnology
Name –Shubham Shriram Wagh
Reg.no - 2017BTGT070
Visited place - KVK
(Paithan road , Aurangabad)
Date of Visit - 26-10 -2021

1. KVK(Aurangabad) is one of the best govt. Institution that provides the training programs to the Farmers &

students.

2. It is established in 1983.

3. The Area of institute is 50 Acres.

4. It also play an important role in transfer of

agriculture technology under University .

 AVAILABLE FACILITIES
• Soil testing laboratory - This service provide at reasonable rate for farmers.
• Agricultural Exhibition - Once in a year, the exhibition is arranged by institute.
• Hydroponics techniques- Soilless Farming project are under working.
• Water Testing Laboratory
• Biofertilizer
Functions
1. Krishi Vigyaan Kendra arranges the various need based training programs for farmers.
2. They gives knowledge of advanced rural farming technology to the farmer.
3. The KVK,Aurangabad act as a chain link between University and farmers.
4. It supplies varieties of breeds of various goats and poultry farmers.
REFERENCES
1.Keb-Llanes, M., Gonzales, G., Chi-Manzanero, B. and Infante, D. 2002. A rapid and simple method for small-scale DNA
extraction in Agavaceae and other tropical plants. Plant Molecular Biology Reporter 20(3):299.
2.Voytas, D., 2000. Agarose gel electrophoresis. Current protocols in molecular biology, 51(1), pp.2-5.
3.Murasnige, T. and Skoog, F., 1962. A revised medium for rapid growth and bio agsays with tohaoco tissue cultures. Physiol.
plant, 15(3), pp.473-497.
4.Khare, S.R., Kharate, P.S., kumar Sahu, R. and Jha, Z., 2021. The Rapid in-vitro micropropagation of Bamboo (Dendrocalamus
strictus) and its genetic fidelity testing using ISSR markers. Environment Conservation Journal.
5.Kumar, S.R., Ram, K.S., Kharate Pawankumar, S. and Zenu, J., 2021. Clonal fidelity assessment of in vitro propagated
Bambusa balcooa plant using DNA marker. Research Journal of Biotechnology Vol, 16, p.4.
6.Tripathi, D.M., Gautam, D., Tiwari, D., Ahuja, D., Sharma, D.A. and Singh, D.A., Introduction to Bioinformatics (Practical
Manual-MBB 555).
7.Rohlf, F.J., 1992. NTSYS-pc: numerical taxonomy and multivariate analysis system. Applied Biostatistics.
8.de Albuquerque, E.M.B., Almeida, F.D.A.C., Gomes, J.P., Alves, N.M.C. and da Silva, W.P., 2015. Production of “peanut
milk” based beverages enriched with umbu and guava pulps. Journal of the Saudi Society of Agricultural Sciences, 14(1), pp.61-
67.
9.Diarra, K., Nong, Z.G. and Jie, C., 2005. Peanut milk and peanut milk based products production: a review. Critical reviews in
food science and nutrition, 45(5), pp.405-423.
10.Snyder, H.E. and Wilson, L.A., 2003. SOY (SOYA) BEANS| Processing for the Food Industry.