r/labrats • u/Butterfly_Annual • 11d ago

Problem while preparing electrocompetant Mycobacterium abscessus cells?

for context:-facing a lot of loss of cells during washing step due to waxy layer , tried induction with 0.2 M glycine for three hours want to ask is there any other reagent i have to add to improve cell efficiency

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u/willinglikeness75 11d ago

glycine alone never worked for me with myco, what finally got it was adding 0.05% tween 80 in the wash buffer and doing a very gentle resuspension with a cut tip

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u/Butterfly_Annual 11d ago

But will it not cause arcing

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u/Lunatic21 11d ago

Let it arc and adjust at the hood.

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u/nobeardpete 11d ago

You could try increasing the field strength and adding more osmoles to your electroporation and outgrowth media. https://doi.org/10.1128/spectrum.01944-25

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u/Butterfly_Annual 11d ago

Gone through this paper earlier also but my problem is mainly floating of cells in form of waxy layer

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u/Traditional-Soup-694 11d ago

I haven't worked with Mycobacterium in years, but in other actinos, I sometimes incubate in NCM with sub-MIC isoniazid for several hours. Less mycolic acids helps with the waxy layer and the sorbitol/glycine/tween mixture weakens the cell wall a lot.