• Question from:Max Berrill

Hello,

I'm currently working out the transformation efficiency for a bacterial transformation with plasmid DNA. My value is an order of magnitude lower than the expected values provided by Invitrogen.

I have a few ideas which I feel comfortable explaining (sub-competent instead of competent cells & handling errors). However, in the troubleshooting table on the Invitrogen website it states that an excess in mass/volume of DNA may result in lower transformation efficiencies than expected (1-10ng pDNA). In my experiment I used 20ng of DNA so this may be a source of the lower efficiency, however I'm not sure why?

Invitrogen state that the volume of DNA may cause the decrease, would this be because there would be a large volume of the solution that the DNA is stored in, which would interfere with the transformation?

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  • University of Bristol
  • Posts: 4,230

at high concentrations DNA is directly toxic to competent cells. As well if you dilute the reaction mix too much then that will also reduce the efficiency of transformation since there is less chance the DNA comes in contact with the competent cells.

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  • University of Bristol
  • Posts: 560

Ah Max!! To follow on from David at high [DNA] you also could have an increased amount of 'impurities' (salts, etc) that can affect transformation efficiencies. Do you really think that you can accurately compare the stated Invitrogen effciency using a plasmid (pUC19) prepared by whatever method (how much is supercoiled DNA?) with your plasmid sample? Also, look at the Invitrogen spec sheet - did you follow it exactly?

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  • University of Auckland, New Zealand
  • Posts: 464

I take a very dim view of the claimed competancies of commercial cells, especially electrocompetent cells. These are very sensitive to changes in temperature, which can happen all too easily during shipping or during the transformation.

But realistically, unless you are making very large libraries, you don't really need super-high competencies. In single cloning experiements, using home-made cells, I have quiet happily settled for ~10^5 cfu/ug. Just plate out a bit more of the transformation!

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  • University of Bristol
  • Posts: 560

Very much agree with you John! Most commercial suppliers give a 'quality control' of 'up to' a certain number of transformants anyway, and this doesn't mean much when you are transforming something that you have sub-cloned (especially with a difficult ligation). It's a pity that the art of making your own competent cells has been largely lost - dito constructing libraries!