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电转化的一些技巧与经验

原文链接:https://international.neb.com/tools-and-resources/usage-guidelines/electroporation-tips

Electroporation Tip

1. Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice.

2. Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes.

3. Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency.

4. 对于连接反应,可以用乙醇沉淀DNA,而后用水溶解以去除盐离子。Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than 10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns are recommended for clean up of ligation reactions.

5. Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap.

6. Arcing may occur due to high concentration of salts or air bubbles.

7. It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.

8. 预热琼脂板可以提高转化效率。Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.

9. 对于未使用完的感受态细胞,可以重新冷冻。Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.

10. 对于Bio-rad Micropulser, EC1 的出厂设置条件是1.8 kV,通常用于0.1 cm电转杯;EC2的设置是2.5 kV,常用于0.2cm 的电转杯;EC3的设置是3.0 kV。

11. 对于E. coli, 最佳的电转化条件是使用0.2 cm电转杯,40 uL 细胞,2.5 kV,时间常数(time constant)约5 ms

12. 对于酵母S. cerevisiae, 最佳的电转化条件是0.2 cm电转杯,40 uL细胞,1.5 kV电压,时间常数是5 ms

 

作者: 于浩然

本科及硕士毕业于天津大学,博士毕业于伦敦大学学院。现就职于浙江大学化学工程与生物工程学院,PI,博导。研究方向为蛋白质工程、生物催化剂、合成生物学等。



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