Hi all,
I am trying to extract DNA from Alternaria spores. They prove to be very tough to open. Does anyone have a lab protocol they are using to obtain a high yield of Alternaria spore DNA? Currently, I am using the E.Z.N.A. Universal Pathogen Kit and bead milling with a range of bead sizes from 0.1-0.5 mm. I am thinking of trying a pre-DNA extraction step of incubating with lyticase overnight at 30C, or trying a freeze thaw step with LN2.
Any help would be much appreciated!
Cheers,
Melissa
Hi Melissa: Why you don't try to extract DNA from fungus mycelium is too easy?
I think use the lysis kit that have enzymatic lysis for spore in the better option for you
If you want to quantify fungal spores, why do you not count them with an optical microscope as real spores from a known volume and with a grid instead of with the more expensive and laborious technique of qPCR? DNA methods are not the answer to everything. Simple is best.
I have used PrepMan™ Ultra Sample Preparation Reagent, however from fungus mycelium.
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Hi Mel Larrabee
There are several references on attachment 1
https://www.ncbi.nlm.nih.gov/pubmed/10445315
6
https://norgenbiotek.com/product/fungiyeast-genomic-dna-isolation-kit
8
https://onlinelibrary.wiley.com/doi/10.1046/j.1365-3059.1999.00406.x/pdf
9
https://onlinelibrary.wiley.com/doi/10.1046/j.1365-3059.1999.00406.x/pdf
14
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102915
g
http://130.226.173.223/euroblight/Workshop/2013Limassol/Proceedings/Page189-194_Leiminger.pdf
m
https://onlinelibrary.wiley.com/doi/10.1046/j.1365-3059.1999.00406.x/pdf
Dear Mel Larabee,
You can try to use CTAB method to perform DNA extraction of your spores. This method is suitable for extraction of plant tissue which its cell wall is quiet tough to break. You can also use Nitrogen Liquid to grind your spores before the extraction procedure.
Best of luck.
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