Preparation for Mother plates

Preparation of Media

  1. Nutrient Agar for Bacterial identification
  2. Potato Dextrose Agar for Fungal identification
  • Prepare NA and PDA medium and dissolved in 500 ml Distilled water
  • Autoclave the prepared media at 121 degree Celsius for 15 min.
  • After 30 mins media keep in laminar air flow under UV treatment for 10min it help to prevent contamination.

Plates Preparation

  • pour the plates
  • wait until media gets solidify
  • After the solidified media treat the plates under UV for 10 mins
  • After solidification Agriculture compost culture were inoculated by spread plate technique.

After 5 days unfortunately on both medias contamination were observed.

Again Same process were followed with the proper care and handling.

  • After 2 days again contamination were observed on NA plate
  • After 8 days various colonies of fungus were observed on PDA.
  • This PDA plate are preserve as a mother culture in refrigerator.

Sub culturing of mother plates

  • PDA plates were prepared For first sub culturing
  • single colony were took and strike on PDA by four quadrant method.
  • we had about 8 different colonies with different color and shape .
  • All these colonies were separately grown in different PDA plates. And incubated a 37°C for 7-8 days.
  • After 8 days pure colonies were observed. These are also preserved for second sub culturing.

Second Sub-Culturing

Same process as above were followed

Third Sub-Culturing

same process as above were followed

After proper growth of fungi i.e. after formation of spores, We have Lysed the cell by using liquid nitrogen.

  1. Firstly we scaped the growth by using sterile spatula, and then took it in clean mortal.
  2. Added Liquid nitrogen to mortal and steriring it to form powder of fungs.
  3. This powder is then used to Extarct DNA for Identification of perticular fungi.

DNA EXTRACTION PROTOCOL

(Reference; Aamir S. Sutar S. Singh SK, Baghela A. 2015. A rapid and efficient method of fungal genomic DNA extraction, suitable for PCR based molecular methods. Plant Pathology & Quarantine: 5 (2): 74-81)

Take DNA extraction tube & add approx. 70 mg glass beads

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Then add approx. 300mg of fungal mycelium in DNA extraction tube

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Add approx. 1 ml of lysis buffer and homogenize it at 6M/S for 60s twice

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Centrifuge at 10,000 rpm for 10 min and separate supernatant in new Eppendorf tube

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Add equal volume of Phenol: Chloroform:Isoamylalcohol (PCI ) and shake well, Centrifuge at 10,000 rpm for 10min.

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Separate upper aqueous layer in new eppendorf tube and add equal volume of Chloroform: Isoamyl alcohol (CI).

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mix well by inverting the tube

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Centrifuge at 10,000 epty for 10 min, separate upper aqueous layer in new eppendorf tube

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Add equal volume of Isopropanol and keep at -20°C for 20 min (100% ethanol)

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Centrifuge at 10,000 rpm for 10 min at 4°C Remove supernatant and wash the pellet using 500 ul of 70% ethanol.

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Again centrifuge at 10,000 rpm for 5 min at 4°C Remove supernatant and dry the pellet

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Dissolve the pellet in 50-70 ul , of IX TE buffer

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Add 1ul of RNase and incubate it at 37°C for 30 min

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2ul of genomic DNA is subjected to 0.8% agarose gel electrophoresis

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then observe the gel under UV trans illuminator gel documentation system

Bands of DNA

Functions of Components used in DNA Extraction

Phenol: Chloroform: Isoamyl alcohol

Nucleic acid solutions commonly contain undesirable contaminants that are chiefly made of proteins. The nucleic acid solution is extracted by successively washing with a volume of phenol (pH 8.0): a volume of phenol: chloroform; isoamyl alcohol (25: 24: 1) and chloroform: isoamyl alcohol (24;1). The contaminants are denatured and accumulate in the organic phase or in the marginal layer between the two phases and the nucleic acids are preserved in the aqueous phase

Isopropanol

DNA is insoluble in alcohol; it will aggregate together, giving a pellet upon centrifugation. Isopropanol is used for precipitation of DNA.

70% Ethanol

To remove salts and other water soluble impurities.

Buffers: TAE, TBE, TE

Composition: 50X TAE (Tris-Acetate-EDTA) for IL

242 g of Tris base

57.1 ml Glacial acetic acid

100 ml 0.5 M EDTA (pH 8)

Make final volume to 11. using dH,O

Composition: 50X TBE (Tris-Borate-EDTA) for IL

242 g of Tris base

137.5 g of Boric acid

100 ml 0.5 M EDTA (pH 8)

Make final volume to IL using dH₂O

Composition: IX TE (Tris EDTA) for 10ml.

0.1 ml of IM TrisHCH (pH 8)

0.02 ml. 0.5 M EDTA (pH 8)

Make final volume to 10 ml. using dH,O

Function:

TAE/TBE buffer provides an ionic solution that allows current to pass through the water. The Tris is useful in keeping the DNA deprotonated. EDTA protects nucleic acids from degradation. It removes certain metal ions from enzymes that would destroy the nucleic acid, thus inactivating the enzymes.