Hi Mr. Burby,First, I would like to thanks you for this very...

RA
Raquel Azevedo
Feb 20, 2019
protocol Protocol: CRISPR/Cas9 Editing of the Bacillus subtilis Genome
Hi Mr. Burby,

First, I would like to thanks you for this very helpful protocol. I am now doing the Gibson reaction and transformation of MC1061 competent cells. I get colonies after transformation but with no way to check if the plasmid was correctly assembled and if it is in. I
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Tobias Zumbusch Answered Jul 5, 2019

Fachhochschule Aachen

Hello Raquel,

how many clones did you pick for screening regarding the spacer integration?

As soon as i remember it's "low-copy" plasmid meaning you will only get around 50 ng/ul plasmid for a standard plasmid mini prep kit. so instead of using just 2 ml try to use 4ml ore more for the initial cell harvest, perform a bit variation regarding lysozyme and use for the final elution step not 50 ul maybe 30 ul. That will be still enough for further cloning procedures.

I'm also focussing on B. licheniformis but since i purified more than 100 clones and spacer integration was never succesful i switched to another plasmid.

Best
Tobias Zumbusch
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