Hello,

We wish to electroporate ES cells with protein nickase D10A and two sgRNAs targeting our gene of interest.

Before adding the RNP complexes to the electroporation cuvette containing our cells :

Should the RNP complexes be formed independently? (1 tube with nickase D10A + sgRNA1 and 1 tube with nickase D10A + sgRNA-2) or mix the 2 sgRNAs and the nickase? (1 tube with nickase D10A + sgRNA1 + sgRNA2).

What concentrations/volumes do you use to form the RNP complexes prior to addition to the electroporation cuvette.

How many ES cells do you electroporate with RNP complexes?

Thank you

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