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transformation efficiency


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#1 turtle

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Posted 08 April 2004 - 11:19 AM

Hi. I have a question about the transformation efficiency of competent E. coli. I know that the efficiency is calculated based on the amount of plasmid used in the transformation and the nuber of colones resulting. Does the initial volume of competent cells affect this at all? The maufacturer claims that using less than 100 ul of competent cells in my transformation procedure will reduce the efficiency of transformation. I don't understand this - if I also reduce the amount of plasmid I use, shouldn't the efficiency stay the same?

Does the efficiency calculation assume that every cell takes up a plasmid?

If my cells have an efficiency of < 10^9 cfu/ng can I just use more cells to start with and end up with a comperable level of colonies?
Thanks for your help.

#2 tfitzwater

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Posted 14 April 2004 - 06:33 AM

The volume and the type of tube used both have effects on the transformation efficiency. This is because the final temperature the cells reach during the heat pulse is affected by these parameters. If the final temperature is too high, the cells die. If the final temperature is too low, the transformation efficiency is reduced. This was published in the early 80s in a BRL Focus article. I have successfully transformed cells with a plasmid containing a temperature-induced promoter construct by pulsing at 30C for 5 minutes. But I don't know if that time point was optimal.

The cells take up more than one plasmid and, in addition, the cell surface is coated with other copies of the plasmid (as well as excess copies of the insert DNA fragment since the ratio of insert to vector is usually greater than 1:1) that did not get taken up.

Invitrogen and Statagene have both published brochures on transformation efficiency and the number of colonies required to generate a cDNA library.

Calcium chloride protocols typically generate 5e6 to 2e7 cfu/ug of supercoiled plasmid DNA or 5000-10,000 cfu/ng of ligated DNA.
The Hanahan protocol generates 5e8 cfu/ug of supercoiled plasmid DNA or 500,000 cfu/ng of ligated DNA.
Electroporation protocols generate 1e9 to 1e10 cfu/ug of supercoiled plasmid DNA or 5,000,000-10,000,000 cfu/ng of ligated DNA.

There is a plasmid saturation level effect also. The cfu/ng plateau is surprisingly low, but will vary acording to the type of competency and the cell strain.

Edited by tfitzwater, 14 April 2004 - 06:33 AM.


#3 turtle

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Posted 14 April 2004 - 08:58 AM

Thanks. If I understood your answer correctly, I don't have to worry too much about optimal efficiency if I am merely cloning for sequencing, rather than building libraries.

#4 Antigen

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Posted 19 April 2004 - 07:10 AM

Manufacturer would be glad you to use 100 and more ul of cells! ))
I use 10 ul cells and 1 ul of plasmid for recloning perposes. Works always!

#5 hula

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Posted 19 April 2004 - 08:57 AM

Manufacturer would be glad you to use 100 and more ul of cells! ))
I use 10 ul cells and 1 ul of plasmid for recloning perposes. Works always!

Good point!




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