Dna extraction

extract fungal DNA (Rhizoctonia solani)

1. The way to the genetic material

2. Cell wall
Plasma membrane
Nucleus
The genetic material (DNA) Other organelles
Disruption
Purification
Cytoplasm

3. DNA extraction components
1.Detergent: such as cetyl trimethyl ammonium bromide (CTAB) which
disrupts the membranes
2.Chelating agent: such as EDTA which chelates the magnesium ions
required for DNase activity
3.Buffer : which is almost always Tris at pH 8 and a salt such as
sodium chloride which aids in precipitation by neutralizing the
negative charges on the DNA so that the molecules can come together.
4. Chloroform: isoamyl alcohol (24:1), to remove protein
5.Isopropanol : for DNA precipitation.
6.Ethanol: removes salts and other alcohols

4. DNA extraction protocol

5. 1.Grind mycelium with cetyltrimethylammonium bromide CTAB
buffer (CTAB, Tris-HCL, ethylenediaminetetraacetic acid
(EDTA), and NaCL) using mortar and pistil.
2.Transferred grinded mycelium into 1.5 mL eppendorf, then
vortexe thoroughly and incubate them at 60°C for 30 min.
3. Add cold chloroform: isoamyl alcohol (24:1), mix well and spin
the samples at 9,000 rpm for 15 min.
4.Add cold isopropanol to the supernatant in a new eppendorf tube
and mix well, then keep at -18°C for 30 min. in order to
precipitate the DNA.
5.Centrifuge at 14,000 rpm for 15 min.
6.Wash the precipitated DNA with 70% ethanol and left to dry in
a laminar flow cabinet.
7.Re-suspended the optained DNA in 50 μL of TE buffer (Tris-HCL
pH 8.0 and EDTA).