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士鋒生物裂褶菌DNA提取方法
最近更新時間：2013-11-12
提 供 商：上海士鋒生物科技有限公司資料大?。?/span>13.6KB
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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 .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
.5 g MgSO4
20 g Glucose
10 g Agar
.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
.5 g MgSO4
20 g Glucose