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This method is a convenient way to purify high-quality genomic DNA from yeast cells. It is suitable for PCR and other assays that require genomic DNA of higher quality.

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[Bio101] Yeast Genomic DNA Miniprep Using A FastPrep Cell Lyser
[Bio101] 使用FastPrep快速细胞裂解仪微量制备酵母基因组DNA

分子生物学 > DNA > DNA 提取
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
5/5/2011, 7069 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.64

[Abstract] This method is a convenient way to purify high-quality genomic DNA from yeast cells. It is suitable for PCR and other assays that require genomic DNA of higher quality.
Keywords: Genomic DNA(基因组DNA), Yeast(酵母), Small scale(小尺度), FastPrep Cell Lyser(FastPrep 细胞裂解仪), PCR(PCR)

[Abstract] 该方法是从酵母细胞中提取高质量的基因组DNA的简便方法,比较适合用于PCR或其他分析所需的高质量基因组DNA 的提取。

 

Materials and Reagents

  1. 5 M Ammonium acetate (pH 7.0)
  2. Chloroform
  3. Isopropanol
  4. 70% Ethanol
  5. Lysis buffer (see Recipes)

Equipment

  1. Adapted for Fastprep machine
  2. Screw-tube
  3. Glass beads
  4. Microfuge

Procedure

  1. Grow 5 ml yeast cells overnight at 30 °C.
  2. Spin,wash once with 1 ml H2O.
  3. Resuspend in 500 μl lysis buffer.
  4. Transfer to a screw-tube with acid washed glass beads.
  5. Fastprep at 6.0 speed for 2 min.
  6. Recover liquid phase with blue tip into another tube.
  7. Add 385 μl 5 M ammonium acetate pH 7.0.
  8. Incubate 5 min at 65 °C, then 5 min on ice.
  9. Add 500 μl chloroform, vortex, spin 2 min in microfuge.
  10. Take supernatant and precipitate with 1 ml isopropanol.
  11. Incubate 5 min at room temprature, then spin 5 min.
  12. Wash pellet with 70% ethanol, dry and dissolve in 50 μl H2O.
    Note: For Southern, digest 5 μl DNA; For PCR, use 0.5-1 μl DNA. For E coli transformation, use 1-5 μl DNA.

Recipes

  1. Lysis buffer
    100 mM Tris (pH 8.0)
    50 mM EDTA
    1% SDS
    For 50 ml: 5 ml 1 M Tris, 5 ml 0.5 M EDTA, 5 ml 10% SDS
 

材料和试剂

  1. 5 M乙酸铵(pH 7.0)
  2. 氯仿
  3. 异丙醇
  4. 70%乙醇
  5. 裂解缓冲液(见配方)

设备

  1. 适应Fastprep机器
  2. 螺杆管
  3. 玻璃珠
  4. Microfuge

程序

  1. 在30℃下培养5ml的酵母细胞过夜。
  2. 旋转,用1ml H 2 O洗涤一次。
  3. 重悬于500μl裂解缓冲液中。
  4. 转移到带有酸洗玻璃珠的螺旋管。
  5. Fastprep在6.0速度2分钟。
  6. 用蓝色尖端将液相回收到另一个管中。
  7. 加入385μl5M乙酸铵pH 7.0。
  8. 在65℃孵育5分钟,然后在冰上孵育5分钟。
  9. 加入500μl氯仿,涡旋,在微量离心机中旋转2分钟。
  10. 取上清液并用1ml异丙醇沉淀。
  11. 在室温孵育5分钟,然后旋转5分钟。
  12. 用70%乙醇洗涤沉淀,干燥并溶解在50μlH 2 O中 注意:对于Southern,消化5μlDNA; 对于PCR,使用0.5-1μlDNA。 对于大肠杆菌转化,使用1-5μlDNA。

食谱

  1. 裂解缓冲液
    100mM Tris(pH8.0) 50mM EDTA
    1%SDS
    对于50ml:5ml 1M Tris,5ml 0.5M EDTA,5ml 10%SDS
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How to cite this protocol: Li, X. (2011). Yeast Genomic DNA Miniprep Using A FastPrep Cell Lyser. Bio-protocol Bio101: e64. DOI: 10.21769/BioProtoc.64; Full Text



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10/10/2013 6:09:42 AM  

cecilia ojesola
Federal university of agriculture, abeokuta

can i use sodium or potassium acetate in place of ammonium acetate?.

10/10/2013 9:58:30 PM  

Xiyan Li (Author)
Department of Genetics, Stanford University, USA

Potassium is almost replaceable with ammonium as cations. The difference here is the pH. NH4Ac is naturally at pH7.0 without any adjustment. But you have to add acid to KAc for pH7.0, which changes the overall concentration of acetate.
Besides, ammonium acetate is volatile, thus "cleaner" than potassium for downstream applications.

Xiyan

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2/28/2013 7:25:52 AM  

Awesome protocol! It also works for Actinomycetes...

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9/3/2012 5:33:30 PM  

Hats off to whoever wrote this up and posetd it.

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