Hello Natalia,

I will answer the questions you mentioned in order.

1. Use liquid culture or fungal samples from plates.

I would recommended that you use fungal samples obtained from liquid culture medium. Fungi in this state are more active, generally have reached their logarithmic growth phase, have thinner cell walls, and have higher enzymatic efficiency. If fungi are obtained on solid culture medium, they may be more likely to cause mechanical damage during collection and cause DNA shearing.

When collecting, avoid enrichment by centrifugation. You can use sterilized gauze to filter and enrich fungal precipitates.

2. Can the sample be freeze-dried?

It is not recommended to use freeze-dried samples for the same reason as above. After freeze-drying, fungal samples may not be fresh enough and are not easy to be enzymatically hydrolyzed. It is best to extract DNA after fresh sampling, or to extract DNA after liquid nitrogen quick freezing.

When extracting, it is recommended to digest the cell wall first. Here we recommend our digestive enzyme Zymolyase (HY-E70306) (optimum temperature 30℃). After digestion, protoplasts can be obtained. Lysis and DNA extraction at this time can avoid DNA shearing caused by excessive use of mechanical methods to damage the cell wall. In addition, the lysis temperature should not exceed 65℃ (high temperature for more than 1h will cause DNA depurination and affect the nanopore sequencing signal)

3. How to choose between mortar and magnetic bead method.

It is recommended to use magnetic bead method. It is emphasized that traditional phenol-chloroform extraction must be avoided, which may cause DNA breakage. When adding liquid after lysis, you cannot mediate, and you need to hold the centrifuge tube and gently turn it up and down. Similarly, the control parameters must be controlled during centrifugation (< 1,000 g).

In terms of consumables, it is recommended to use a wide-mouth gun tip.

Please let me know if you have other question.