Genomic DNA Isolation

Raise fungal mycelium in liquid culture medium

(Malt extract- 10g l-1; Glucose- 5g l-1). Take a small piece (approx 100 mg) from the liquid culture and dry it by removing excess water by pressing with pipette tip.

Crush the mycelial culture in tubes in liquid nitrogen (use special sticks for tubes).
Use Quagen kit for DNA isolation. Add Qiagen lyses buffer AP1 400l/ vial.

Add RNase A 4l/tube which is maintained at 4C on ice. Incubate at 65 C for 10 min (keep mixing by inverting the tubes).

Add 130 l AP 2 Buffer and keep for 5 min on ice.

Transfer in Q1 Ashredder Mini Column (Pink).

Centrifuge for 2 min at 14000 rpm.

Collect supernatant and discard pellet.

Add 675 l buffer AP 3/E.

Transfer 650 l in DNeasy Mini Column (white).

Centrifuge at 9000 rpm for 1 min.

Discard liquid of outer column and add the rest of 675 l in the inner column.

 Centrifuge again at 9000 rpm for 1 min.

Discard liquid from outer vial and add 500 l buffer AW in inner column. Centrifuge again at 12000 rpm for 2 min. Discard liquid of outer tube and add once again buffer AW @500l/tube. Dry the columns by tissue paper and put each tube back in new tubes and add 50l buffer AE (Elusion buffer has to be maintained at 60 oC for 10 min).

 Centrifuge at 9000 rpm for 1 min (due to action of elusion buffer AE the DNA will now be dissolved and pass through the column). Do not disturb DNA of the outer tube, add again 50l of elusion buffer AE to the inner column.

Incubate it for 5 min, centrifuge it again at 9000 rpm for 1min(DNA Purified).

DNA Quantification
Use DNA fluorimeter - HoeferDyna Quant 200 Calibration of DNA fluorimeter
Put on the switch

The equipment is run automatically. Let it stabilize for 15 min.

Press1> read Take 2 ml of Assay Solution A in cuvette Insert cuvette and press zero

Display will show 0 ng ml-1

Add 2 ml of standard calf thymus DNA solution (100 ng ml-1)

Press calibration.

Enter standard value (100 ng)

Screen will display 100 ng ml-1

DNA quantification of unknown sample

Take 2 ml assay solution A Press enter two times

Press zero (Display 0 ng ml-1)

Add 2 ml DNA sample to assay solution and mix by pipetting Press enter and quantify DNA in ng ml-1 of DNA solution.

 Preparation of solution

Assay Solution A (Low range, 10-500 ng ml-1 final

DNA concentration) 10X TNE buffer Hoechest dye stock solution (1mg ml-1)
Preparation of solution

10 ml 10 ml 90 ml

dH2O

Hoechest dye stock solution (1mg ml-1) Hoechest dye dH2O Store at 4C in amber colour vials. 2 mg 2 ml

 10 X TNE buffer
100mM Tris base 12.11 g

10mM
2M

EDTA
NaCl

3.72 g
116.89 g

Dissolve in 800 ml of water. Adjust pH to 7.4 with

concentrated HCl and raise the volume to 1000 ml with

dH2O. Filter before use. Store at 4C up to three months.