I'm isolating DNA from frozen cell pellets at 2e7 for a half-library screen using HEK293T cells.
My issue is that from each 2e7 pellet I am obtaining approximately 45-60 ug, while given a 6 pg/cell estimation I should have ~110-120ug.
I am fairly certain that the cell count is correct, as all cells were re-seeded to flask...
I want to delete a gene using a Transient CRISPR method. I have few queries regarding gene deletion:
1\. I want to use single gRNA with high off and on target score. How efficient it could be or I can use 2 gRNAs for better efficiency.
2\. I want to use 3 stop codons followed by restriction site and marker gene. Is there ...
We recently prepared to use SAM for screening experiments, but we found that the lentivirus titer of the package was always very low, and the cells were almost dead after adding puromycin. In addition, another CRISPRa library we used (Calabrese, from john doench lab) also had such a problem. Other lentiviruses we packaged h...
I'm going to use gecko library in order to infect primary macrophages and is the very first time I'm approaching this technique, so I have some questions.
I'm following the nat prot paper by Julia Joung (2017) but I have some questions regarding the titration part.
I'm using the 2 vectors system and I will infect macropha...
What do you think, why can be a promoter being transcriptionally silent? If it is silent, do to that there is no native transcriptional activator binding to this promoter, could this be supplemented with a well-guided dCas9-activator?
I was hoping if someone could help me with a simple question. I really tried finding a clear answer to this online but I don't see a defined solution anywhere. This is my question:
I am transfecting HEK293T cells with LentiCRISPRv2 + ssDNA via Lipofectamine 2000 in a 12 well plate. I added puromycin after 24 hours of selec...
I am using LentiCRISPR v2 to make a point mutation in HEK293T cells. I have a few general questions:
1\. Is there a defined protocol to do HDR with LentiCRISPR v2?
2\. I transduced my HEK293T cells with the LentiCRISPR v2 cassette that had my gRNA integrated into it. I saw a specific cut on the site S421 (on the huntingti...
I did a literature search tailored to find ways to increase HDR in Human IPS - ES cells. I was interested in trying drugs to enhance Homology directed repair.
Has anyone has tried any of the following drugs- constructs in their lab
or know of any which I have left off the list.
I will be introducing a SNP into an IPSC ...
I packed lentivirus from HEK293T cell line with lenticas9-blast #52962. I started selection with blasticidin 3 days post infection. Control group were dead and the infected cells were selected over one week. But it took at least one week to recover the cells (had to withdraw Blast) to have a decent number of cells to run WB...

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