This is a method for low-cost electrotransformation of electrocompetent NEB turbo E coli cells using a homemade electroporation apparatus.
The original paper upon which this method is based can be found here: https://doi.org/10.1371/journal.pbio.3000589
My method for preparing electrocompetent E coli can be found here: h...
I compared the results from identical cloning protocols preceding either a heat-shock or electroporation bacterial transformation into NEB competent E. coli cells (chemically competent or electrocompetent) and found that the electroporation transformation consistently resulted in thousands more colonies than the heat-shock ...
I am new to CRISPR and I am interested in HDR for now. I was wondering if anyone has already tried using the Cas9-DN1S plasmid from Jayavaradhan et al. (2019) for HDR-CRISPR. I am trying to transfect rat myoblast cells (L6), however its transfection using lipofectamine seems to be low efficient.
I have previously transfect...
We want to create a mouse with a fluorescent tag of an endogenous gene. I would really appreciate advice with recommended CRISPR methods and services.
It seems from the literature the typical method for germline editing is to modify zygotes directly. Is this what everyone would currently recommend?
A colleague who did CRI...
I am following Joung et al. for custom library preparation. I plated cells as recommended 1 electroporation per 500cm dish. 12 hours after incubation, I removed the plates from the incubator. Fortunately, the reaction went well but unfortunately, cells are too dense. There is barely any space between colonies.
Do you think...
I have some troubles in clone my custom library, but I'm not sure what's wrong.
My custom library contains 12000 sgRNAs, which is small. but when we performed the NGS after plasmids extraction, it showed that only 1000 sgRNA was detected, and among the 1000 sgRNA, 300 sgRNA was abundant while others was only 1~2 copy.
1\...
I want to design my own custom library but I have some very basic
questions before starting:
1) How do you obtain your sgRNA? I mean, after you design the guides
is there a company that puts the two oligos together and sends to you
the guide ready to use? So you "just" have to clone it into the
plasmid (lenti guide puro in...
I have a question regarding the quantity of the pooled CRISPR library that should be electroporated in the Endura electrocompetent cells.
According to other posts in this forum, a suggestion was to use 1.5 uL from Addgene library of 50ng/uL for each electroporation, that is 75ng.
Can this quantity of 75ng be applied as a ...
I tried several approaches including lipofection using CRISPRMax/RNAiMax and also electroporation for delivering RNP of Cpf1 but all failed to be detected in T7EI assay, basically, we just wanted to find the gRNA which works
Does anybody have good experience in KO lncRNA using electroporation of mouse zygotes?
I would very much like to learn from experience re strategy please.

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