Hello! I'm trying to create cell lines of HepG2 with separate knock-outs of mirna 101, 93, 30. I need to separately lower the level of mature miR 101, 93 and 30 in my cells. For this purpose I use CRISPR-Cas system utilising CRISPRMAX, TrueCut HiFi Cas9 protein and gRNAs. After transfection I conduct PCR from these genes and then cut these fragments with mismatch endonuclease I by BioLabs. I supposed to get smaller fragments after restriction, however I get only larger fragments in my agarose gel (they are above fragments on which restriction wasn't performed) . Does it mean that my transfection is unsuccessful? And how can I improve the result of my transfection? Last time I plated cells only the day before conducting the transfection and the cell density was approximately 40-50%.
Hi Kristina,
it seems transfection is not successful. Have you used any positive control that contains some tag like GFP, which can be visible to demonstrate successful transfection? Cell density should be higher for transfection if you follow the electroporation technique.
Sangeeta Kumari, thank you for your answer! I used control such as non-effective gRNA plus cas9, so theoretically under good conditions in this case there wouldn't be any mismatched base pairs and the amount of miR will be the same as in the wild cells.
I used such low density because I found articles in which it was mentioned that low cell density is crucial for effective transfection of cells.
I can't understand how I can use positive control using tag-GFP in this case because I try do create a deletion not insertion.
If you preform your PCR on untreated cells do you get the correct band size? If not it may be an issue of your primers not amplifying the correct locus. Do you know the copy number of your region of interest in HepG2 many cell lines have more than 2 copies of each chromosome making it harder to get a complete knockout.
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