By knowing the mechanism and determining factors, we will know how to increase the transformation efficiency in microbes.
Hi Yijie,
I hope I understand your question correctly. If I do I can give a rather basic overview. Firstly, during the preparation of competent cells, chemical treatments are expected to alter the cell surface charge, in order to improve DNA interaction with the cells under cold conditions. The heat shock step is expected to destabilise the membrane and increase its permeability, allowing entry of DNA. The cells are then replaced on ice to allow the membrane to restore, followed by recovery in nutritional medium (e.g. SOC).
Rather uncomplicated in other words. From my experience, the critical step for transformation efficiency lies in the competent cell preparation, so I would recommend starting there for optimisation. Otherwise you can play around with incubation times of the transformation, and ensure that adequate quality and quantity of DNA is being added.
I hope this is helpful!
Kind regards,
Chris
It is generally thought that heat shock can create pores on cell membrane and the ions like Ca2+ neutrolize the negative charges of the membrane and DNA. Together with other mechanisms, these help DNA entry into bacteria.
Yes, pretty much. Calcium ions or certain other positively charged ions. Sometimes additional chemicals can increase the permeability further.
Good luck with your experiments...
Hi! I asume you are doing transformation with heat shock method and not with good results, you can try electroporation instead, but remember always make the cell membrane leaky Good luck
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