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Electrocompetent cells and PCR products do not mix

Electrocompetent cells and PCR products do not mix - Molecular Cloning Forum

Electrocompetent cells and PCR products do not mix - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 05-07-2009, 08:32 PM
Pipette Filler
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Default Electrocompetent cells and PCR products do not mix



I'm trying to modify a bacterial artificial chromosome and to do so, need to prepare electrocompetent cells, mix with a PCR product (designed to undergo homologous recombination with the BAC) and electroporate. Immediately upon addition of the PCR product, the bacterial suspension gets expelled up the walls of the eppendorf tube. Subsequent electroporation, recovery and plating of such samples never produces any colonies. This expulsion phenotype seems to be independent of the bacterial preparation: when several electrocompetent samples are prepared simultaneously, addition of the exact same PCR product causes expulsion in some eppendorf tubes but not others.

The electrocompetent bacteria are suspended in water and the PCR products are also suspended in water. Any ideas what might be causing this and how to fix it?
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bac , cells , electrocompetent , mix , pcr , products


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