biological mutagenesis - (Feb/19/2007 )

hi everyone
i m using mutant e.coli strains to induce mutations in a plasmid i inserted
but the rate of mutations seems very very low
does anyone know why? how can i improve it?

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you could hydroxylamine to induce random mutations, from memory it deaminates adenine to hypoxanthine that prefers to bind to cytosine hence at to gc transitions.

need to incubated with your plasmid for different time periods and transform your bugs and analyse


hope this helps

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probably your selection system is bad.
you can use a simple PCR amplification (with Taq polymerase) on that plasmid. the TaqPol inserts many mutations. (this method is called RAM)
then insert the plasmids to the cells, and do your selection. afterward sequence the plasmids of the selected colonies.

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