Dear all, I am a rookie in mycology. I am trying to extract DNA from cultured mycelium. However, I have failed to obtain good concentration (less than 5 ng/uL from 50 uL elution mixture) and DNA purity measured by nanodrop is not promising as well, 260/280 reading was about 1.4 to 1.7; while 260/230 was less than 1 most of the time.
I have repeated the washing step twice and also added 1 step 70% ethanol washing. It didnt seem to improve the readings. Elution with water instead of elution buffer did improve the 260/230 reading slightly, yet still less than desirable (~1.4).
I am wondering what else could I try to improve my DNA concentration and purity. It seems to be affecting my downstream processes.
Here are some information for consideration:
1. DNA extraction kit tried:
- QIAGEN's DNeasy blood and tissue
- ZYMO's Quick-DNA Fungal/Bacterial Miniprep Kit
2. Volume of elution buffer used = 50 uL
3. Partial agar was collected during mycelium harvesting
Thank you.
I suggest to reduce the elution volume and increase the elution time for a more concentrated eppendorf.
Try to minimize the agar amount for a better DNA quality
Dear Ahmed Ahmed Adel Gereisha , noted, will try out your suggestions. Thank you.
You should concentrate on steps of digestion/hydrolysis of mycelium. You can vortex more upto more 1_2 minutes.
Dear Ashok Ashok Kumar , thanks for your reply, the kit i am using has bashing beads, i let it bash in the homogeniser for about 15 minutes for the initial step. Doesnt seem to improve the reading significantly. Should i incubate overnight instead?
I think you should increase the cycles of homogeniser and also control the temperature fluctuations. If you are using enzymes in kit, then add little more units and incubate more minutes.
Dear Ashok Ashok Kumar , noted on more cycles and temperature fluctuations. I'll give it a try, thank you!
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