I am trying to isolate the plasmid from E. coli BL21 DE3 in an expression host. The concentration of plasmid DNA is high if I isolate it with boil lysis method but the yield is poor.
Another problem is that if I use isolated plasmid DNA for restriction it is getting fully digested i.e. no band, only smear is appearing on the gel.
Can anyone help me in removing this nuclease activity?
Re-transform DH5-alpha or DH10B (top ten), which are endonuclase free strains, with your plasmid, let them grow overnight, and isolate your plasmid again which will be suitable for the restriction analysis.
EdgeBio has a cell line called Acella that is a BL21(DE3) mutant that has complete deletions of recA and endA that is appropriate for both plasmid propagation and expression.
recA and endA are the two enzymes in E. coli that frequently mess with your plasmid and are deleted in DH5-alpha or other common strains used for this purpose, but the Acella cells combine the benefits of an expression strain with a plasmid isolation strain.
http://www.edgebio.com/products/acella%E2%84%A2-chemically-competent-cells-tubes-36795
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