Hi,
I am considering to buy MAX Efficiency DH5 alpha Competent Cells to try to do both electroporation and heat-shock experiments with large plasmids. I don't know what procedure will be better, so I am planning to try both of them.I am just getting familiar with this kind of stuff , so this is a completely new field. I am aware that people prepare his own stock of competent cells. I have a few questions about that.
If I want to prepare my own stock of electrocompetent cells from these MAX Efficiency DH5 alpha Competent Cells bought, I streak them from the vial provided on a LB plate and I follow a protocol of mine to make them electrocompetent..is it right?But, do the cells keep their characteristics to be at max efficiency in this way? I mean, I would not use directly the vial provided but the cells I grow by myself.Would they keep the same features to be at high efficiency for the transformation with large plasmids? This is valid also if I want to prepare my own stock of chemically competent cells?
Does the protocol they provide with the cells refer to their direct use or can it also be applied to my own stock of competent cells(after streaking and preparation of competent cells)?
https://tools.thermofisher.com/content/sfs/manuals/18258012.pdf
Hey there,
The competency of a given stock of electro or chemically competent cells only arises by preparing them that way, not from any intrinsic competency of the cells themselves (although some E. coli strains do have better transformation efficiencies than others). So if you streak the cells onto a fresh agar plate they will no longer be competent and will need to be made so again. If you do this then make them competent yourself they are unlikely to be as competent as they were before. If you buy competent cells you should only defrost them when you need to use them and keep them as cold as possible throughout (until the heat shock, in the case of chemically competent cells).
How large is the plasmid you would like to transform? If you have lots of it I would try making your own chemically competent cells from a lab stock of DH5a cells or something. If you are really struggling then making or buying some electrocompetent cells (much more competent than chemically competent cells) may be the way to go. You could use that protocol for your own cells too.
Hope that helps!
Hey there,
The competency of a given stock of electro or chemically competent cells only arises by preparing them that way, not from any intrinsic competency of the cells themselves (although some E. coli strains do have better transformation efficiencies than others). So if you streak the cells onto a fresh agar plate they will no longer be competent and will need to be made so again. If you do this then make them competent yourself they are unlikely to be as competent as they were before. If you buy competent cells you should only defrost them when you need to use them and keep them as cold as possible throughout (until the heat shock, in the case of chemically competent cells).
How large is the plasmid you would like to transform? If you have lots of it I would try making your own chemically competent cells from a lab stock of DH5a cells or something. If you are really struggling then making or buying some electrocompetent cells (much more competent than chemically competent cells) may be the way to go. You could use that protocol for your own cells too.
Hope that helps!
Rory is quite correct, when you make competent cells (either for chemical or electroporation) yourself then the any strain of DH5a is going to be much the same. There is nothing magical about the strain used for the MAX efficiency, the magic is just that the company is really good at quality control in making competent cells.
If you need high efficiency then you are better off using commercial cells, if you can survive with normal efficiency for routine cloning, then you can probably make them yourself if you are careful and test your batch for quality. Electroporation is normally quite a bit more efficient than transformation, but it really depends upon your needs.
Keep in mind; on freezing competent cells lose competency over time. If you prepare your own competent cells you can save them in liquid nitrogen. This will keep your cell competent more time than the ones kept in -80oC. As mentioned earlier, competent cells are treated cells and lose competency on subculturing.
Pretty much all the comments raised here are true, but the cells you received are probably as good as you are going to get transformation wise. If you want high competency then use them straight from the vial but bugs grown from the stock you bought can grown up and make them competent. I would also bear in mind the answer from Salah and aliquot them and freeze them quickly in liquid nitrogen and don't keep them for too long, and batch test to assertion their transformation efficiency. When I moved to a lab with a bit more cash I used bought in competent bugs as they were always better, but got good results from the ones I made myself in the past.
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