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tranformation trouble using dh5alpha


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#1 laborat

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Posted 12 January 2011 - 10:22 PM

I would be helped if anyone can provide an insight into using dh5alpha competent cells to transform a ligated plasmid. The plasmid size is 12kb with insert. one of my labmate could transform the same plasmid of 12kb in the dh5alpha cells but I have had many failures with this. what is the maximum size of plasmid that this competent cell can take up. your suggestions are highly appreciated.

#2 HomeBrew

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Posted 13 January 2011 - 03:16 AM

I don't know what the maximum size is, but DH5-alpha can easily accommodate a 12 kb plasmid, so your problems are not due to the size of your plasmid...

Can you give us more details on what you're doing, like:

-- Are you transforming from a ligation reaction, or trying to transform with a whole, purified plasmid?

-- If you're transforming from a ligation mixture, can you tell us how you prepared your vector, how you prepared your insert fragment, and what ligation protocol you used?

-- What is your vector and how are you selecting for transformants?

-- How were your competent cells prepared, and what controls have you done to insure they're competent?

-- When you say your "labmate could transform the same plasmid of 12kb", do you mean exactly the same plasmid (vector + insert), or just another plasmid of 12 kb? Is your labmate using the same batch of competent cells?




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