How to Extract DNA of Rust Fungi Colonized on Plant Matter

Step-by-Step Guide

1. 1

   Step 1: Add to Lysing Matrix C tubes 25-25mg infected leaf tissue (thumbtack size) and 20-25mg diatomaceous earth (a scoop of a toothpick)

   Invert every 15 minutes to mix.,, Invert to mix.,, Discard previous tube., Invert to mix.,,, Invert to mix.
   
   If white precipitate does not form, add an addition 50μL,, Discard previous tube.,,, You may want to arrange tubes in the same direction, so the pellet is always on the same side after centrifugation., Pour off the supernatant, being very, very careful not to dislodge the pellet of DNA.
   
   If there is no pellet, you need to re-attempt the extraction., If the pellet does not go into solution on its own, incubate for 5 minutes at 37 °C (99 °F)., You may wish to evaluate the quality/quantity of the DNA on an agarose gel.
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   Step 2: Place samples in MP FastPrep 24 homogenizer for 20s on speed 4.

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   Step 3: Add 600μL Genomic Lysis Buffer with Proteinase K. Quickly

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   Step 4: vortex to completely mix.

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   Step 5: Incubate at 60 °C (140 °F) for 1 hour.

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   Step 6: Let cool to room temperature.

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   Step 7: Add 200μL Chloroform.

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   Step 8: Spin at highest speed for 10 minutes.

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   Step 9: Transfer supernatant (top layer) to a clean 1.5mL tube.

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    Step 10: Add 50μL DNA Stripping Solution.

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    Step 11: Incubate at 60 °C (140 °F) for 1 hour.

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    Step 12: Quickly

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    Step 13: place on ice for 5 minutes.

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    Step 14: Add 100μL Precipitation Solution.

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    Step 15: Spin at highest speed for 5 minutes.

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    Step 16: Without disturbing the pellet

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    Step 17: transfer supernatant to a clean 1.5mL tube.

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    Step 18: Add 5μL Mussel Glycogen and 500μL Isopropanol.

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    Step 19: Invert at least 50 times to mix.

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    Step 20: Spin at highest speed for 10 minutes.

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    Step 21: There should be a small pellet at the bottom.

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    Step 22: Add 100μL TE Buffer to the pellet.

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    Step 23: DNA extraction is complete.

Detailed Guide

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