To begin, transfer 20 milliliters of the fermented grape sample into a sterile 50 milliliter screwed cap tube. Then add 10 milliliters of cold water into the tube and vortex for homogenization. Centrifuge the sample at 800 G for one minute at four degrees Celsius.
Transfer the supernatant to a new 50 milliliter tube and centrifuge at 3000 G for 20 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in one milliliter of PBS. Transfer the cell suspension to a two milliliter screw cap tube and centrifuge at 14, 000 G for two minutes.
Now resuspend the pellet in 978 microliters of sodium phosphate buffer of pH 7.4 and 122 microliters of DNA buffer. Vortex the tube and place it at four degrees Celsius for 45 minutes to one hour. Transfer one milliliter of the sample into a lysis solution one tube and tighten the cap.
Homogenize the sample three times in a bead beating grinder for 60 seconds each. Then centrifuge the lysing matrix E tube for one minute at 16, 800 G.Transfer the supernannt into a clean microtube and add 250 microliters of protein precipitation solution to the tube. Shake the tube 10 times to mix the contents.
Centrifuge the sample for five minutes at 16, 800 G and transfer the supernatant into a sterile 15 milliliter tube. Then add one milliliter of binding matrix suspension to the supernatant and mix by inverting the tubes for two minutes before placing them in rack for three minutes. After removing one milliliter of the supernatant, resuspend the matrix in the remaining supernatant.
Transfer 600 microliters of the mixture into a spin filter tube and centrifuge. Then decant the flow through and add 500 microliters of wash solution into the spin filter tube. Remove the spin filter and place it in a fresh catch tube for five minutes.
After drying, add 50 microliters of warm DNAse-free water on the filter membrane and gently stir the matrix with a pipette tip. Centrifuge at 14, 500 G for one minute to elute the DNA. Load the sample onto an agarose gel.
Finally, measure the DNA concentration.
