CRISPR-Cas9–mediated knockout was performed using an established methodology (38). The short guide RNA (sgRNA) sequence (5′-AAATTTGTCAGCAACTCGTC-3′) from the GeCKO library (39) was cloned into the Bbs I restriction site of the SpCas9-2A-Puro V2.0 vector, which was a gift from F. Zhang (Addgene plasmid no. 62988). MDA-MB-231 cells were transiently transfected with the sgRNA plasmid, grown for 48 hours, and selected with puromycin (0.5 μg/ml; Invitrogen). Surviving cells were plated as single-cell colonies in a 96-well plate (1 cell per well) and expanded. Knockout of TRPM7 in expanded cells was confirmed by Western blotting with an anti-TRPM7 antibody (clone N74/25, Abcam).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.