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Extraction of DNA from Schizophyllum commune (裂褶菌DNA提取)

标签: 裂褶菌 DNA提取

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1. Transfer plugs of mycelial margin to 0.1 P plates. Allow to grow to fill plate (about one week at 21°).

2. Macerate two plates of mycelia in a blender with 50 ml of .002 P liquid medium. Inoculate 50 ml flasks of .002 P liquid medium with 4 ml each of this macerate. Shake at 150 rpm for one week at room temperature. (See Niederpruem, D.J.; Marshall, C.; Speth, J.L. Control of extracellular slime accumulation in monokaryons and resultant dikaryons of Schizophyllum commune. Sabouraudia 15: 283-295; 1977.)

3. Mycelia may be filtered from these cultures through miracloth and washed 4x as in step 5. Yield is about 50 mg of lyophilized tissue per 50 ml culture, i.e. 1mg/ml.

4. One 50 ml culture may be used to inoculate 1000 ml of 0.002 P medium in a 2L Fernbach flask, and shaken for 1 week at room temperature.

5. Filter mycelia in miracloth, squeeze out liquid, resuspend in ddH2O, and stir a few minutes. Repeat to a total of 4 washings. Squeeze excess water from mycelia and freeze in -70.

6. Lyophilize mycelia.

7. Grind mycelia under liquid N2 in mortar (prechill mortar in -70).

8. Mycelia may be stored dessicated (with a miracloth bag of drierite in a ziplock bag) in the -20.

9. Extract DNA using the Coprinus procedure (modified from Murray and Thompson. Nucleic Acids Res. 8:4321-4325; 1980.)

0.1 P agar

For 1 L total medium:

5 ml Phosphate stock

1 ml trace elements stock

5 ml thiamine HCl

1 g L-asparagine

0.5 g MgSO4

20 g Glucose

10 g Agar

0.002 P liquid medium

For 1 L total medium:

100 ml Phosphate stock

1 ml trace elements stock

5 ml thiamine HCl

1 g L-asparagine

0.5 g MgSO4

20 g Glucose

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