Kasson Lab DNA Extraction
Angie Macias, Matthew T Kasson, Brian Lovett
Abstract
This is a routine protocol for extracting DNA from various fungi. This extraction method is suitable for follow-up molecular work such as PCR amplification.
Steps
Before you begin
Turn on hot water bath, set to 65°C.
Pull two Eppendorf 1.5mL centrifuge tubes per sample.
Label both sets of tubes with (short) sample names.
Label one tube set for each sample with an "I" for
<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.q26g78413lwz/Screen%20Shot%202020-10-28%20at%201.12.23%20PM.png" alt="Sketch of "I"-labeled tubes (Angie Macias)." loading="lazy" title="Sketch of "I"-labeled tubes (Angie Macias)."/>
Add 600µL of
Add 600µL of
Place tube with 65°C water bath.
Extraction Protocol
Sterilize some metal scrapers with flame and 95% (v/v)
Add 1/2 pea-sized amount of fungal tissue (young hyphae) to each tube containing
Flame-sterilize and cool scrapers between samples.
Macerate each sample with a new, sterile micropestle until tissue is homogenous.
Add tubes to a floating rack to allow samples to incubate directly in 65°C water bath for 0h 30m 0s.
Remove samples and vortex for 0h 0m 3s before returning to 65°C water bath for 0h 30m 0s.
Place a sufficient aliquot of
Remove samples and allow them to cool on the bench for 0h 5m 0s.
Add 200µL of
Centrifuge samples for 0h 3m 0s at 14.000rpm,0h 0m 0s.
Using a P1000 micropipette, transfer supernatant to each tube containing
Centrifuge for 0h 1m 0s at 14.000rpm,0h 0m 0s.
Carefully pour off the supernatant into waste container.
Add 600µL of 70% (v/v)
Centrifuge for 0h 1m 0s at 14.000rpm,0h 0m 0s.
Repeat Step 16.
Open and invert tubes onto a clean paper towel.
Add 100µL of warmed
Store fully-labeled tubes in a box (not a tube rack) in the -20°C freezer.
