how to clone unstable DNA? - (Apr/22/2008 )

hi
which is the best way of cloning an unstable DNA (I mean linearized lentiviral plasmid DNA after ligation)?
any commercial competent E coli is OK?
thank you inadvance

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Hi,
We use DH5alpha. It works fine. Competent E coli can be easily prepared.

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hi WHR, I am using TOP10 preparing by Calcium Chloride
but this competent cell only works to my Control plasmid
does not work to unstable plasmid
should I change my competent cell
the competency of my TOP10 is about only 10⁴~10⁵
commercial competent cell is at least 10⁸

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For unstable cloning I would recommend the Sure (Stratagene) or STBL2 (Invitrogen) strains of bacteria. You will never achieve decent transformation efficiency with CaCl2 preparation. Try the CCMB80 approach here:
http://openwetware.org/wiki/TOP10_chemically_competent_cells

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thank you pahage434
I find the reason why my control plasmid transformation gave colonies and ligation product did not
I appreciate your kind help, phage434

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