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MycoMap

First Trial - Microwave Extraction

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Trying out some new extraction techniques. My standard extraction protocol takes to long when processing samples in bulk, so I needed to look for some more efficient methods. I believe I found it with the following protocol. ITS1F & ITS4 as primers.

Primary reference: 

Fast microwave-based DNA extraction from vegetative Mycelium and fruiting body tissues of Agaricomycetes for PCR amplification

Dörnte and Kües DNA.pdf

Protocol:

  1. Take a small piece of tissue from the interior flesh of a mushroom. Place the tissue in a 1.5 uL eppi tube that contains 100 uL of water or TE Buffer.
  2. Microwave the eppi tube for 1 minute.
  3. Let the tube cool to room temperature. (About 30 minutes)
  4. Put the tube in the -20 freezer for 10 minutes.
  5. Centrifuge at 9300g for 5 minutes.
  6. Utilize 10 uL of the supernatant as the template for a PCR reaction.


While testing this method, I made several simple alterations to the protocol in additional trials.

A. Add 100 uL of 99% Isopropanol. Centrifuge for 2 minutes at 16g.

X. Add 100 uL of water. Vortex for 10 seconds. 

Y. Add 100 uL of EtOH. Invert the tube 5 times to mix. Centrifuge 1 minute. Remove all EtOH. Let open tube dry completely upside down on Kimwipe. Add 40 uL of water. 

H: Put tube in dry heat bath for 30 minutes.

The reason I added the extra Isopropanol and EtOH steps was to wash the nuclei lysis solution from the sample and tube. I guessed that the high level of detergents would inhibit PCR, and that proved to be a correct assumption. The heat bath step was to see if that would further aid extraction.


Results:

Lane 1: Ladder
Lane 2: Standard protocol - TE Buffer + H 
Lane 3: Standard protocol - TE Buffer + HAX
Lane 4: Standard Protocol - TE Buffer
Lane 5: Standard Protocol - TE Buffer 
Lane 6: Standard Protocol - Nuclei Lysis Solution
Lane 7: Standard Protocol - Nuclei Lysis Solution + AY
Lane 8: Standard Protocol - Nuclei Lysis Solution + HAX
Lane 9: Standard Protocol - Nuclei Lysis Solution + HAY
Lane 10: No sample

microwave-trial-1.jpg

As a reference I ran a series containing my standard extraction protocol on the bottom. The brightest band in the microwave test series was the closest to the steps I use in my standard protocol. That also seems to be the best series of steps when using the microwave extraction. The additional 30 minutes in the heat bath in lanes 2 and 3 also seemed to be beneficial. But the straightup microwave to PCR supernatant produced nice clear bands in lanes 4 and 5, so the additional heating, while improving the extraction, may be superfluous. It works on a broad range of species. Rather than controlling for taxonomic group, I ran different species with each trial. This shows all methods work across a broad range of genera. All tissue was taken from fresh specimens.

Lane 2: Hygrocybe flavescens.  
Lane 3: Xeromphalina tenuipes
Lane 4: Psathyrella sp.
Lane 5: Sarcoscypha sp.
Lane 6: Mycena leaiana
Lane 7: Rhizomarasmius pyrrocephalus
Lane 8: Trichoglossum sp.
Lane 9: Artomyces pyxidatus

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