Rapid isolation of yeast genomic DNA: Bust n' Grab.
Rapid isolation of yeast genomic DNA: Bust n' Grab.
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DOI：
10.1186/1472-6750-4-8
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发表时间：
2004-04-21
影响因子：
3.5
通讯作者：
Peterson KR
中科院分区：
文献类型：
--
作者：
Harju S;Fedosyuk H;Peterson KR
酵母人工染色体（YAC）的诱变通常需要分析大量酵母克隆以获得正确靶向的突变体。分离酵母基因组DNA的常规方法利用玻璃珠或酶消化来破坏酵母细胞壁。使用小玻璃珠是混乱的，而当需要分析许多样品时，细胞的酶消化是昂贵的。我们试图开发一种比现有方法更容易和更快的方案，用于从平板上的液体培养物或菌落中获得酵母基因组DNA。在裂解缓冲液中重复冻融细胞用于破坏细胞并释放基因组DNA。细胞裂解后，用氯仿提取DNA并进行乙醇沉淀。从1.5ml过夜液体培养物或从大菌落中可以分离出200 ng - 3 μg基因组DNA。将样品直接重悬于限制性酶/RNA酶混合物中用于Southern印迹杂交或用于几个PCR反应。我们通过显示对含有诱变的人β-珠蛋白基因座YAC的酵母克隆的分析来证明该方法的实用性。开发了一种从液体培养物或直接从菌落中获得酵母基因组DNA的有效、廉价的方法。该方案避免了酶或玻璃珠的使用，因此在处理大量样品时更便宜且更容易执行。
Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.
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影响因子：
6.2
作者：
Peterson, Kenneth R
通讯作者：
Peterson, Kenneth R
影响因子：
56.9
作者：
STRAUSS, WM;DAUSMAN, J;JAENISCH, R
通讯作者：
JAENISCH, R
影响因子：
64.8
作者：
JAKOBOVITS, A;MOORE, AL;KLAPHOLZ, S
通讯作者：
KLAPHOLZ, S
影响因子：
2.6
作者：
Le, YZ;Sauer, B
通讯作者：
Sauer, B
DOI：
10.1073/pnas.76.3.1035
发表时间：
1979-01-01
影响因子：
11.1
作者：
STRUHL, K;STINCHCOMB, DT;DAVIS, RW
通讯作者：
DAVIS, RW