Isolation of plant genomic DNA

1. Isolation of Genomic DNA
SAYALI MAGAR
Ph.D. Research scholar
(AGRICULTURAL BIO-TECHNOLOGY)
Dr. P.D.K.V. Akola

2. Principle
The isolation of DNA usually begins with the lysis of cells or tissues in
order to destroy the protein structures and allows the release of
nucleic acids from the nucleus

3. Reagents
1. Extraction(CTAB) Buffer 1.4 M NaCl; 100 mM Tris (pH 8.0); 20 mM
EDTA (pH 8.0) 2% Mercaptoethanol and 2% CTAB
2. Isopropanol
3. Chloroform : isoamylalcohol ( 24:1) mixture
4. Tris:EDTA ( 10mM:1mM) pH 8.0 10 mM Tris
5. 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.
6. 70% ethanol

4. Miscellaneous
1. Mortar and pestle
2. Pipettes and sterile tips
3. Sterile centrifuge and microcentrifuge tubes
4. Sterile glassware

5. What are the Most Commonly used DNA
Extraction Procedures?
1. Organic (Phenol-Chloroform) Extraction
2. Non-Organic (Proteinase K and Salting out)
3. Chelex (Ion Exchange Resin) Extraction
4. FTA Paper (Collection, Storage, and Isolation)
5. Silica Based (Silica exchange resin- Qiagen)

6. ORGANIC EXTRACTION
Perhaps the most basic of all procedures in forensic molecular biology is the
purification of DNA. The key step, the removal of proteins, can often be
carried out simply by extracting aqueous solutions of nucleic acids with phenol
and/or chloroform

7. Procedure
Weigh 2 g of clean young leaf tissue and grind to fine powder with a pestle and mortar after
freezing in liquid nitrogen.
Transfer to 2 ml centrifuge tube with 1.5 ml extraction buffer maintained at 65° C in a water
bath. Mix vigorously or vortex
Incubate at 65° C for one hour. Mix intermittently. Allow to come to room temperature
Centrifuge at 10,000 rpm for 15 min
Take supernatant 600𝜇𝑙 and transfer to a fresh centrifuge tube
Add equal amount of chloroform : isoamyl alcohol. Mix gently
Centrifuge at 10,000 rpm for 10 min

8. Transfer aqueous phase to a fresh centrifuge tube. Add equal volume of chilled isopropanol and let
the DNA to precipitate for 30 min. (by keeping it in – 20° C deep freezer)
Centrifuge at 10,000 rpm for 10 min
Drain out the excess chemicals with a pipette
Add 200 − 300𝜇𝑙 of chilled 70% ethanol
Spin at 10,000 rpm for 5 min
Decant off and dry the pellet under vacuum or air dry
Dissolve DNA in 30𝜇𝑙 of TE
Add RNAse A ((10mg/ml) and incubate at 37° C for one hour
Store eluted DNA at 4 C for frequent use or -20°C for long-term storage.

9. ORGANIC EXTRACTION
REAGENTS
Cell Lysis Buffer - Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer
cell membrane of cells, but will not break down nuclear membrane.
EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent
cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is
bound up by EDTA, nucleases are inactivated
Proteinase K - it is usual to remove most of the protein by digesting with
proteolytic enzymes such as Pronase or proteinase K, which are active against a
broad spectrum of native proteins, before extracting with organic solvents.

10. Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to
extract once with phenol, This procedure takes advantage of the fact that deproteinization is
more efficient when two different organic solvents are used instead of one.
Phenol is highly corrosive and can cause severe burns.
Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl
alcohol is added to help prevent foaming.
The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1

11. Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA.
Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is
smaller.
Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces;
moreover, solutes such sodium chloride are more easily coprecipitated with DNA when
isopropanol is used.

12. DNA quantification
Nanophotometer
Agarose Gel Electrophoresis