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Dna extraction from melia Dubia plant,SlideShare for dna extraction,ppt presentation of extraction of DNA

1. Project report on
Estimation of outcrossing rates in Melia Dubia Cav . Using Simple
Sequence Repeat (SSR) Marker
Submitted by- Under the Supervision of
Anu Kumari Dr. Aditya Kumar( Scientist E)
Department- Msc .Biotechnology G&TI Division
Roll no- 23NMBT596307 IFP, Ranchi, 834001
Registration no- 20VCBSBT066
Session- 2023-2025

2. INTRODUCTION OF MELIA DUBIA
Melia dubia popularly known as Malabar Neem is a promising multipurpose tree
highly suitable for farm forestry and agro forestry for generating higher income
per unit area . Melia Dubia is one such alternative species suitable for timber,
plywood, pulpwood, and fuel wood. More than 80% of the world`s population
presently uses herbal medicines for their primary health care as alternative
system of medicine .
Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Spindales

Family : Meliaceace

Genus : Melia

Species : Dubia

Bionomial Name : Melia Dubia Cav.

3. Simple Sequence Repeats (SSR): SSRs or microsatellites are short
tandem repeats of 1-6 bp in which dinucleotide repeats (NN)n are
most common.
SSRs are abundant and mostly evenly distributed across the genome .
In SSR markers, microsatellite locus is amplified using specific primer
pair from unique sequence flanking the microsatellite locus and
length polymorphism is detected by electrophoresis. SSRs are
co-dominant markers in which polymorphism is due to varying
number of repeats resulting in length polymorphism.

4. CHEMICAL REQUIRED FOR DNA EXTRACTION
1.C TAB buffer
2.NACL(Sodium Chloride)
3.TRIS HCL
4.PVP(Polyvinyl pyrrolidone)
5.EDTA(Ethylene diaminetetra acetic acid
6.βMercaptoethanol

5. WORK OF CHEMICAL
1.CTAB Buffer:- Cationic detergent ,which remove polysacharide and
used for break down the cell membrane.
2.NACL:- used for DNA precipitation.
3.EDTA:-remove all magnesium ion and prevent from degradation of
DNA.
4.TRIS HCL:- maintain PH after cell brust.
5.PVP:-absorb polyphenol
6.Βmercaptoethanol:-strong reducing agent, which clean tannis and
other polyphenol present in plant extract.
7.Chloroform: Isoamylalcohol:- antiforming agent , which remove
lipids.

6. MATERIAL AND METHOD
Selection of mother- The mother and progeny seed sample are collected
from the IFP field , Gutwa, ranchi.

7. SEED COLLECTION AND MEDIA PREPARATION
3.Preparation of media:- media consists of a 2:1 mix of soil and sand enriched with
organic amendments .

4. Seed sowing: : Plant pre-treated seeds in porous sandy loam soil, either in seed
trays or raised nursery beds. Germination typically begins within 7–10 days.
1.Collection of seed: seeds are collected from the ripened fruits by rubbing,
Seeds are collected from IFP (institute of Forest Productivity Ranchi).

2.Seed treatment- Soaking: Soaking in water for 24 hours.
Depulping
Drying
5. Seed Germination
temperature- 32 °C
Humidity- 70-85

9. DNA ISOLATION PROTOCOL

1. 100-200mg leaf sample was grinded in motor pestle with the help of
extraction
buffer(1ml).
2. Sample was taken in 2ml micro centrifuge tube and left in water bath for 1
hr at 65°C.
3. Add equal volume of chloroform- isoamylalcohol (24:1) after some time of
water bath and
mix them.
4. Centrifuge at 12000 rpm, for 14 minute at 24 °C and repeat this one`s
again.
5. Supernatant was taken in another tube.
6. Add 500ml isopropanol and mix them.
7. Store at -20°C at overnight.
8. Again centrifuge at 4°C, 12000rpm for 14 minute.
9. Discard supernatant .
10. Then DNA pellets was washed with 500ml diluents .Centrifuge at
12000rpm , for 12 minute at 24°C.
11. Discard supernatant and left for air dry at room temperature (2-3 hours).
12. Dissolved in 100ml of TE Buffer (Tris base+ EDTA).

10. Quantitative result of extracted DNA
Nanospectrometery:-



1.Distilled water was added to the cuvette as a blank to set nanospectrometer.
2.The genomic DNA was diluted with TE – buffer in the cuvette.
3.The absorbance was measured to both 260 and 280nm to check the purity of DNA.
4.The concentration of DNA is noted.



The yield of extracted DNA quantity was measured by using a nanodrop
spectrophotometer to calculate the purity of DNA at a ratio of 260/280nm and the
purity of Nucleic Acid at 260/230nm

11. Qualitative Result of Extracted DNA
Electrophoresis is a technique used to separate and sometimes purify
macromolecules especially proteins and nucleic acid that differ in size and
charge. It involves an electric field and molecules are separated by an
electric field through a gel that contain small pores. The molecule travel
through the pores in the gel at a speed that is inversely related to their
length. DNA and RNA are negatively charged molecule they will be
pulled towards the positively charged end of the gel.
Electrophoretic System:-

12. Quantitative result( PCR AMPLIFICATION)
Step of PCR
1.Denaturation
2.Annealing
3.Extension
4.Final extension
Primer used
MAZ 2
MAZ3
MAZ5
MAZ6
MAZ 7
MAZ8
MAZ9

MVO1
MVO2
MVO3
MVO4
MVO5
MVO6
MVO7
Component of PCR
Primer(F/R)
Taq Polymerase
DNTPs(Deoxynucleotide
Triphosphates)
10X buffer +Mgcl
2

Mgcl
2

Molecular water
DNA sample

13. SI.
NO.
PRIMER
NAME
FORWARD PRIMER(F) REVERSE PRIMER(R) PRIMER
LENGTH(bp)
1. MAZ-1 F: AGGAAGAATGCCGCTGACTA R: GGAAATGAAAACCGAAAGCA F: 20,R: 20
2. MAZ-2 F: GGGGAAGAGGGTCCAAGTT R: TGAAAAACAATTATGTGATTTAGAAGA F: 19,R: 27
3. MAZ-3 F: ACAATTGGGGAAGTCTGTGC R: GCGCGAACTTCACTCTCTCT F: 20, R: 20
4. MAZ-4 F: TCGTCATAACGCGAGAGTCA R: CTTCGGCTTCTTCTGATTGG F: 20, R: 20
5. MAZ-5 F: TTCTGGAAAACCAACCAACC R: CCTGAGTAAAAGCTACTCTGAATGG F: 20, R: 25
6. MAZ-6 F: TTAGGCATGGATCACAGAAAA R: CAGATTGCTGCAAATTGGTAAA F: 21, R: 22
7. MAZ-7 F: GGAAAGAGAGAAATGGTGCAA R: GACGCGACTTGAACTCAAAA F: 21,R: 20
8. MAZ-8 F: GGGTGTCTTTGGACGTGATT R: CAACGCATGAAAGAGGAAAA F: 20,R: 20
9. MAZ-9 F: AGCTCGAATCCATCCAGAAC R: CCCTCTCTGTCTCTGACGCTAT F: 20, R: 22
10.MVO-1 F: TGTCTATCCGGTGAAGGTGT R: TTGCATACAATAATATCGTGCAAC F: 20,R: 24
11.MVO-2 F: CCCTATTCATTGTCCCTCCA R: GTCCTCCTGGAATTCTGTGC F: 20,R: 20
12.MVO-3 F: AGAGAAGACAGATCCCCCCAGT R: CAACACAACACAACACAGCAA F: 21,R: 21
13.MVO-4 F: CAACCATGGTGTCGAGAAGA R: TGCTTAATTTGCCTGTGCAT F: 20,R:20
14.MVO-5 F: CTAGACCAGCCCCAAGAACA R: TTCAAGGGCTTCTTCTGAATC F: 20,R:21
15.MVO-6 F: CCAATGTTGTTCAACTATATGAGGTC R: GAATTTCGAAGAGTGCCAAAA F: 26, R: 21
16.MVO-7 F: GGAACCCCAATTTAGGATCT R: TGCTTGGTGAAAACCATAGA F: 20, R: 20

PRIMER USED FOR AMPLIFICATION OF DNA

14. Primer MAZ 2 from sample 1-22 mother-progeny 4

15. RESULT:-
Overall , 31% of mother progeny is heterozygous in nature and rest
are polymorphic and homozygous in nature.
Through our project, 4 mother plant and its 20-20 progeny was
taken.