Hello everyone,
I would like to hear experts' opinions on this matter. I am working on gene knockout using CRISPR-Cas9, but unfortunately, I did not successfully achieve my target gene insertion through bacterial transformation using stlb3.
I am considering purchasing a pre-made plasmid that contains my target gene, specifically product number sc-400082-NIC from Santa Cruz. This product is provided as transfection-ready.
I’m particularly interested in expert opinions on whether using pre-made plasmids can facilitate transfection more easily, and if they generally lead to more successful and efficient results. For the transfection, I will be using HEK293T cells and the CanFast reagent.
Thank you!
Hello Munirah Faleh ,
Short answer: Yes, in general I've made the experience that bought reagents can better than self-made.
From your question it is not clear whether it was not possible to insert your target gene (Cas9?) into your plasmid backbone? Or the knockout you then attempted in 293T did not work?
In general, we achive by far the best DSB/KO effciency by using recombinant Cas9 Protein combined with synthezied sgRNAs (both IDT), and electroporation. If you do not have access to a modern EP device (eg. Nucleofector or Neon), efficiency of RNPs should still be much higher even with lipofection.
There are publications on that - RNPs vs. plasmids.
So, if your considering spending ~500$ on the plasmid, you might as well buy 10µl of Cas9 protein, and have a sgRNA synthesized.
Hope that helps,
Thomas
Hi Thomas,
Thank you so much for your response. To clarify your question: it was not possible for me to insert my target gene into the lentCREISPR v2 plasmid. Therefore, I'm considering purchasing a pre-made plasmid that contains my target gene, sc-400082-NIC, from Santa Cruz.
From your experience can you tell me the estimated time it takes to perform transfection with a pre-made plasmid?
Thank you!
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