The invention discloses a method for rapidly extracting long-fragment
DNA from fresh
fungus mycelia. The method comprises the following steps:
thallus preparation: extracting fresh cultured
fungus mycelia, carrying out
centrifugation to remove
moisture, weighing the mycelia, and rapidly
grinding the mycelia into
powder in
liquid nitrogen;
cell lysis: adding
a DNA extracting
buffer solution, adding60 mu L of 20mg / mL
proteinase K, uniformly mixing, and carrying out water bath at 65 DEG for 30 minutes;
DNA extraction: adding 0.33mL of KAc, carrying out water bath for 20 minutes, carrying out extraction by virtue of a mixture of
phenol,
chloroform and
isoamyl alcohol, carrying out
centrifugation, extracting supernate, adding absolute ethyl
alcohol for
precipitation, standing at a low temperature for 30 minutes, carrying out
centrifugation, and collecting precipitates; purification: adding RNaseA,
processing at 37 DEG C for 30 minutes, respectively extracting by virtue of a mixture of
phenol,
chloroform and isoamylol in a ratio of 25 to 24 to 1 and a mixture of
chloroform and isoamylol in a ratio of 24 to 1, and carrying out low-temperature centrifugation for 5 minutes; and extractingsupernate, adding absolute ethyl
alcohol for
precipitation, selecting precipitates, rinsing, carrying out
air drying, dissolving the precipitates into a
TE buffer solution, and carrying out preservation for later use. The
DNA amount of filamentous fungi extracted by virtue of the method is increased twice, the concentration of the filamentous fungi reaches 277ng / mu L, the lengths of fragments aremore than or equal to 20000bps, and the filamentous fungi are not degraded and are high in purity.