五种毁灭你酵母转染的方式
发表于279 天前 技巧 暂无评论 ⁄ 被围观 1,172+

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酵母DNA转染的方法与E.coli转染的方式十分相似,但是如果不注意细节或者说技巧,同样会导致试验失败。不管你用于做酵母菌双杂合筛选,还是做系统模型,下面列出的几个常见的操作希望能够避免……

1. 忘记加单链DNA

在做E.coli转染是直接使用双链的DNA,而酵母转染需要加入单链“载体DNA”,以强化质粒片段。如果你仅添加你的质粒到转染混合物种,培养三天后,你的平板依然会空空如也。

2. 使用失效的PEG

PEG(聚乙二醇)是酵母转染过程中的关键试剂,但其配制困难而且及其不稳定;制备的PEG的含量是试验成败的关键,失效的PEG溶液因水分蒸发,水/PEG比率发生改变,应该在每次转染时新配制PEG溶液。或者,配制小批量,并用封口膜牢牢密封。

3. 使用处于静默期的细胞

处于对数生长期的细胞可以有效的整个DNA,但也不是说处于静默期的细胞不能用于转染,只是你的转染成功率会低得多。因此最好使用繁殖最活跃的细胞进行转染操作。

4. 使用错误的筛选Marker

这是一个低级的愚蠢错误,但是我不得不承认我就在这个问题上犯过好几次了,双人复核可以有效的降低此类错误,节约你的眼泪。

5. 热击时偷工减料

这个是E.coli转染和酵母转染的最大不同:E.coli相对比较小,仅需要几分钟进行热击即可,但是酵母就不同了,比较耐热击,因此,为了提高转染效率,酵母一般需要热击长达45分钟。我一般都至少采用15分钟,如果低于此转染效率立马下降。如果不着急的话,延长至45分钟,会得到更高的转染效率。

您是否有更好的关于酵母转染的技巧,欢迎你投递给找生物(http://www.91bio.com),或者发送文章至邮箱sharebio[at]gmail.com

以下是原文:

Transforming yeast with DNA is a very similar process to transforming E. coli, but with just enough differences to trip you up if you let your attention slip.  Whether you’re doing a yeast two-hybrid screen, or using yeast as a model system, here are a some mistakes to to avoid…

1. Forgetting to add single stranded DNA

While E. coli readily takes up double-stranded DNA, yeast requires the addition of single-stranded “carrier DNA” to enhance uptake of your plasmid or fragment.  If you only add your plasmid to the transformation mix, chances are you’ll be confronted with a pristinely sterile plate after three days of incubation.

 

2. Using old PEG

PEG (polyethylene glycol) is a crucial ingredient in the yeast transformation buffer.  Unfortunately, it’s annoying to make and goes bad quickly.  The percentage of PEG will make or break the success of your transformation; an old PEG solution has had a chance to evaporate, so that the water to PEG ratio is off.  If you can stand it, make new PEG for every transformation.  Otherwise, make it in small batches, and seal tightly with parafilm between uses to minimize evaporation.

 

3. Using cells in stationary phase

Cells in log phase, or exponential growth, take up DNA with much better efficiency.  This is not to say that cells from a stationary culture can’t be transformed – only that your successful transformation rate will be much lower.  Your best bet is to use cells that are growing rapidly at the time of transformation.

4. Using the wrong selective marker

A stupid mistake, but one that I’ve made more times than I’d like to admit.  Double check to save yourself some tears!

5. Cutting corners when heat-shocking

This is a major difference between E. coli  and yeast transformations: while E. coli is relatively delicate, and requires a heat-shock of less than a minute to take up DNA, the cell wall makes yeast more hardy and resistant to shock.  Thus, for efficient transformation, yeast are generally heat-shocked for up to 45 minutes.  I’ve gotten away with as little as 15 minutes, but any shorter and the transformation efficiency is drastically reduced.  If you’re not in a hurry, let it go the whole 45 minutes, or you’ll get to experience the joy of doing it all over again.

Do you have any tips for a successful yeast transformation?

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