Prediction of PAM sequences All CRISPR arrays were retrieved from CRISPRCasdb, part of CRISPR-Cas++, which can be found at https://crisprcas.i2bc.paris-saclay.fr/ (28). Alignment of CRISPR array spacers to genomes was done via BLAST (29) either programmatically using Spacer2PAM or manually through the web interface. The BL...
Chemically modified oligonucleotide synthesis Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides. Syntheses were performed on an Applied Biosystems 3400 or Expedite DNA Synthesizer at a 1 micromole scale using Unylink CPG support (ChemGenes)...
I would like to delete/mutate a regulatory element in the promoter of a gene in HEK 293T cell lines. However, I have never tried designing guides/donors for knock in/HDR. Can some one recommend me a protocol and vectors/design strategies for this? What are the easiest/most efficient strategies for this at the moment?
I am using Gecko v2 library to screen survival cells after drug treatments. After drug treatments, I have harvested cells and count the cell numbers. As expected, I have 4 times more numbers of cells in the control group (untreated) than in tested groups. I expect that I have more genomic DNA in the control group too. So...
I m approaching to the CRISPR/Cas9 world. I m going to perform an experiment using CRISPR/Cas9 mediated knockouts in primary human T cells from donor PBMC cells and I have some doubts regarding the method. I found two different approaches: the CRISPR/Cas9- mediated gene editing that requires the endonuclease Cas9 and a gu...
I did a gene activation experiment 6 months ago in fibroblasts where I used a guide RNA known to work in HEK293 cells. The guide also worked well in all my experiments using fibroblasts (nucleofection, Lonza Amaxa kit). I am now trying to repeat this overexpression experiment in the same fibroblast cell line but I am not se...
Find your community. Ask questions. Science is better when we troubleshoot together.
Sci Find is a place for experimentation beyond the publication. We are a growing collaborative community of scientists and experimentalists from around the world who believe science is better when we work together.
- Help us keep Sci Find a safe space for productive and collaborative conversations. Please be kind and respect your fellow community members. If you see abusive or alarming content, please report it to firstname.lastname@example.org, and our team will take appropriate actions.
- All information posted to this page is public and Open Access. Learn more about our commitment to Open Access by visiting our Docs.
- We encourage our community to have collaborative troubleshooting discussions, and to share tips and tricks, experimental methods, resources, and opportunities.
- You must be a member to create or engage with content. Sign Up to create your profile and join the community for free.
- Follow a guild and create a post. Learn more about guilds here.
- Post titles should be descriptive and include descriptive tags. Check out our tagging guidelines here.
Have a question?
Contact email@example.com or check out our support page.