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High quality Fungal genomic DNA isolation
Author: Sha Dai, updated date: , view: 58, Q&A: 0

Procedure

用于全基因组测序的真菌基因组DNA的提取

步骤

65℃预热beffer B。准备裂解液:500mg菌体使用17.5ml

利用液氮充分研磨菌体,转移进入50ml液氮遇冷的离心管中,加入17.5ml裂解液,快速涡旋充分混匀。

65℃水浴30min,每5-10分钟轻轻颠倒一次。

加入5.75ml0.33volKAc5M),轻轻颠倒混匀,冰浴30min

4℃,5000g,离心20min

吸取上清到新的50ml离心管内,加入1 vol氯仿:异戊醇:苯酚(25:24:1)萃取,10min4℃,4000g,离心10min

吸取上清到新的50ml离心管内,加入1 vol氯仿:异戊醇(24:1)萃取,10min4℃,4000g,离心10min

吸取上清到新的50ml离心管内,加入130 ul RNase A10mg/ml),37℃水浴2h

加入1/10 vol KAc5M)和1 vol异丙醇,轻轻混匀,室温静止5min4℃,10000g,离心30min

去上清,用70%乙醇清洗,4℃,10000g离心10min

去上清,室温下风干15min左右。

250ul TE,轻弹助融,65℃金属浴2-5min,保存于-80℃。

试剂配制

Buffer A:  0.35M sorbitol (山梨醇)

         0.1M Tris-HClPH9

         5mM EDTAPH8

Buffer B 0.2M Tris-HClPH9

          50mM EDTAPH9

          2M NaCl

          2% CTAB

Buffer C 5% SarkosylN-lauroylsarcosine sodium salt  十二烷基季胺酸钠   

Potassium Acetate 5M KAc precipitate polysaccharides  PH7.5

RNase A 10mg/ml

Proteinase K 20mg/ml  

PVP 1%     聚乙烯吡咯烷酮

ChloroformeIsoamylalcool24:1

Sodium AcetateKAc 3M

Isopropanol 100%   异丙醇

Ethanol 70%

Lysis Buffer        For 17.5ml

2,5 vol of buffer A       6.5ml

2,5 vol of buffer B       6.5ml

1 vol of Buffer C        2.6ml

PVP 1%              1.75ml

Proteinase K           125ul

 

 

 

 

 


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