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DNA Isolation from M. foliorum - any updates?

| posted 13 Oct, 2018 02:47
c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.
| posted 14 Oct, 2018 16:34
c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

I edited my post above to reflect the correct concentration (should someone else refer to it).
| posted 23 Oct, 2018 21:03
Just thought I'd share that we got great DNA concentrations and quality today with our first attempt with this protocol. We only used 2mL (not the 5 our protocol says), and got concentrations around 250ng/ul, with A260/280 of 2-ish. Very pleased! Thanks for all your help! Now to run it on a gel with/without RE buffers…

Nikki
(I've attached our protocol here, in case anyone else wants to try it).
 
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