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Problems with second recombination in mutagenesis with suicide vector

mutgenesis suicide vector second recombination pLD55 counterselection

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#1 Fuza

Fuza

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Posted 17 October 2012 - 04:14 PM

Hello everyone!

I've been using the suicide vector pLD55 for mutagenesis.

The technic consists in integrate the plasmid in the crhomosome by homologous recombination (this step is ready), and then by a second event of recombination obtain a free-plasmid segregant, where the results can be the WT gene again, or the mutation.

Is in this second step where I'm having problems, because sometimes the second recombination doesn't occur at all, or in the case it happens, I only obtain the WT recombinant again

Does anybody knows about a trick behind this? or maybe a recombinase which I could use to enhance the process?

Thanks for the help and sorry for my bad english!





Also tagged with one or more of these keywords: mutgenesis, suicide vector, second recombination, pLD55, counterselection

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