The ‘endo-blue method’ for direct cloning of restriction endonuclease genes inE.coli
- 25 June 1994
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 22 (12) , 2399-2403
- https://doi.org/10.1093/nar/22.12.2399
Abstract
A new E.coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methyla-tion-dependent restriction systems (McrA −; , McrBC − , Mrr −; ). This strain has been used to clone restriction endonuclease genes directly into E.coli . When E.coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes Taq I (5′TCGA3′) and Tth 111I (5′GACNNNGTC3′) have been successfully cloned in E.coli . The new strain will be useful to clone other genes involved in DNA metabolism.Keywords
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