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BioReagent,无菌,用于显微镜 级 ,适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
PEG 是一种高分子聚合物,在酵母感受态转化过程中只有分子量达到 3000 左右的 PEG 才会发挥最大的转化促进作 用。PEG 在酵母转化中起到在高浓度 LiAc 环境中保护细胞膜,减少 LiAc 对细胞膜结构损伤的作用;同时 PEG 可促使质粒更紧密贴附酵母细胞壁。LiAc 可使酵母细胞产生一种短暂的感受性状态,此时酵母能够摄取外源 DNA。PEG/LiAc 是生产的酵母感受态细胞转化所必需的配套试剂,每个转化反应需要 500µl PEG/LiAc。
产品组分与配方:
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操作方法
1、Carrier DNA 的预处理:将 Carrier DNA 插入 95℃ 金属浴 5min 或插入浮漂中 95℃ 水浴 3min,快速插入冰中。
2、取 100µl 冰上融化的酵母感受态细胞,依次加入预冷的目的质粒 2-5 µg,预处理后的 Carrier DNA 10µl,PEG/LiAc 500µl 并吸打几次混匀,30℃ 水浴 30min (15min 时翻转 6-8 次混匀)。
3、将管放 42℃ 水浴 15 min (7.5 min 时翻转 6-8 次混匀)。
4、5000 rpm 离心 40s 弃上清,dd H₂O400µl 重悬,离心 30s 弃上清。 5. ddH₂O或 0.9% NaCl 50 µl 重悬,涂平板。
注意事项:
1、PEG 长时间静置易沉淀,在每次使用前务必摇晃,使试剂中各组分分布均匀。 2、不开封的 PEG/LiAC Solution 可在 4℃ 长时间保存,开封之后最好放-20℃保存,发现污染立即丢弃。
PEG is a high-molecular polymer. During competent transformation of yeast cells, PEG with a molecular weight of approximately 3000 yields the optimal promoting effect on transformation efficiency.In yeast transformation, PEG protects the cell membrane under high-concentration lithium acetate (LiAc) conditions and alleviates structural damage to the cell membrane caused by LiAc. Meanwhile, PEG facilitates tighter adhesion of plasmids to the yeast cell wall.LiAc induces a transient competent state in yeast cells, enabling the cells to take up exogenous DNA.PEG/LiAc Solution is an essential supporting reagent for transformation of prepared yeast competent cells, and 500 µL of PEG/LiAc Solution is required for each single transformation reaction.
Product Composition and Formula:
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Operation Protocol
1. Pretreatment of Carrier DNA: Incubate Carrier DNA at 95 ℃ in a metal bath for 5 min, or in a water bath with a floating rack at 95 ℃ for 3 min, then immediately place it on ice for rapid cooling.
2. Take 100 µL yeast competent cells thawed on ice, sequentially add pre-cooled target plasmid (2–5 µg), 10 µL pretreated Carrier DNA and 500 µL PEG/LiAc Solution. Gently pipette up and down several times to mix thoroughly. Incubate the mixture in a 30 ℃ water bath for 30 min; invert the tube 6–8 times to mix at the 15 min mark.
3. Transfer the tube to a 42 ℃ water bath and incubate for 15 min; invert the tube 6–8 times to mix at the 7.5 min mark.
4. Centrifuge at 5000 rpm for 40 s and discard the supernatant. Resuspend the pellet with 400 µL ddH₂O, then centrifuge for 30 s and remove the supernatant.
5. Resuspend the cell pellet with 50 µL ddH₂O or 0.9% NaCl solution, then spread the suspension onto agar plates.
Precautions:
1. PEG tends to precipitate after long-term static storage. Be sure to shake the reagent thoroughly before each use to homogenize all components.
2. Unopened PEG/LiAc Solution can be stored long-term at 4 ℃. After opening, it is recommended to store at -20 ℃. Discard the reagent immediately once microbial contamination is observed.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
| 分析证书 | P1524607 |
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