nucleofection with Lonza of suspension cells- low efficiency with 10 kb plasmid

I have been working with CRIPSR-Cas system and using nucleofection method for plasmid delivery. I am co-transfecting with10 kb size CRISPR-Cas and 3kb size gRNA using Lonza amaxa 4D-X system. The cells are suspension B cells. Buffers for transfection are company SF buffer and also homemade buffers of which recipes obtained from the literature ( https://doi.org/10.3389/fbioe.2016.00099 ).

The problem is nucleofection with 3 kb size using lonza programs and tested buffers are OK but when I try them with large size plasmid I get max up to 10% ( very bad in my opinion). I need your suggestions, experiences! How do you successfully deliver your plasmids to cells apart from using lentivirus vector. I think this step is the most challenging part. Are there any tips and tricks? Does any of you experience with Lonza transfection or any other nucleofection method? What is your the most optimal transfection efficiency percentage to reach for a good editing efficiency? Any input is welcomed and appreciated!


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Tan Chimeraalmost 2 years ago

Make sure you're recovering your cells in low calcium or zero calcium media for at least 15 minutes and maybe even overnight. Other than that, it's a known fact that larger plasmids have lower transfection efficiency.

Silver Lichabout 1 year ago

Any news on this?

I have also realized that the pmax plasmids do work great, but I think it is due to the size.

When I use the plasmids CRISPR-nCas9-GFP (461, ~10 kb) I get a really low transfection. I am going to use another control, as a labmate has another GFP-cas9 plasmid that has worked before.

Aqua Ghostabout 1 year ago

I think the CBh promoter in these Cas9 plasmids might be not suitable for all cell types. Transfection efficiency is probably not the main issue. Have you ever tried a Cas9 plasmid with a different promoter?

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