Hi everyone, I'm trying to extract fungal DNA from wheat common bunt using chelex100 and I've tried several different protocols without any success. I've used different concentrations of chelex100 (5%, 10%, 20%) with different ways of heating (heat block, water bath, boiling water) and different timings (2x 15 mins, 1x 30 mins plus 1x 15 mins, 2x 20 mins). Only 2 things were fixed in all my tries, 1st thing is that I dissolved chelex100 in double distilled injection water and 2nd thing is that I centrifuged the complex at 13000 rpm for 1 min every time it needed centrifugation. I'm not sure what am doing wrong, Like should I try dissolving chelex100 in any specific buffer or something? or should I change my centrifugation speed and time? or do I need to add anything to the complex in different steps?
I'd appreciate if you take the time and kindly answer my question.
We use Chelex 100 dissolved in TE Buffer (pH8) to isolate DNA from gram positive spore forming anaerobic bacteria. We suspend 1 bacterial colony in 40uL 10% Chelex. Heat at 100C for 15 minutes. Centrifuge at 18000g for 1 minute. Dilute the supernatant 10-fold in TE buffer. DNA suitable for PCR as well as NGS. Hope it helps.
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