Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Biolistic bombardment of Cas9 RNP complexes

MS Manuel Serif
GD Gwendoline Dubois
AF Anne-Laure Finoux
MT Marie-Ange Teste
DJ Denis Jallet
FD Fayza Daboussi
request Request a Protocol
ask Ask a question
Favorite

RNP complexes were assembled following the instructions of IDT and delivered to WT Phaeodactylum cells by particle bombardment. Briefly, 24 h before the experiment, cells (1.5 × 108) were spread onto 1% agar F/2 plates with 20 g L−1 sea salt and 50 µg mL−1 uracil, if targeting PtUMPS. For each shot, the equivalent of 4 or 8 µg Cas9 protein (for multiple targets, the total amount was split equally between the different RNPs) in a total volume of 8 µl Cas9 reaction buffer (NEB, B0386A) was mixed with 10 μl gold nanoparticles (3 mg, 0.6 μm in diameter, Bio-Rad) washed twice with Cas9 reaction buffer. The coated particles were distributed slowly in a circle directly onto the macrocarrier, which was allowed to dry for 2 h before biolistic bombardment. A burst pressure of 1550 psi and a vacuum of 25 Hg were used.

Copyright and License information: The Author(s) ©2018
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A