![]() ![]() until reaching H1, which now has 200 µL of medium. Remove 100 µL from A1 containing cells and mix with B1, pipetting up and down 3x to mix well before removing 100 µL and mixing with C1, etc.(Perform this set-up with at least 2 96-well plates per mutation in order to generate at least 10-20 clones). The other 95 wells should contain 100 µL of the respective medium. Count the cells in order to put ~16,000 cells into the first well (A1) of a 96-well plate containing 200 µL of medium.After the cells have recovered from a technique to induce a mutation, use the serial dilution method as follows: However, it is pretty tedious, and you must work relatively quickly at first. Serial dilution method: This method is exactly as it sounds – you create a serial dilution in order to get down to approximately 1 cell. Below are two methods that I have personally used to successfully generate single cell clones. This sounds difficult, but is actually quite possible! Just a little time consuming. How Do You Generate Single Cell Clones?Ī single cell clone is essentially generated from an original ‘multiclonal’ population, but has been separated from the rest in order to create a pure, clonal population that is genetically identical. However, the cells I generally work with are difficult to transfect (much less than 100% transfection efficiency), so I need to isolate single cell clones from WT clones. And if all you care about is that the message is knocked down, then a western blot looking at protein expression might also suffice. However, after making a mutation, do you know if all of the cells contain the same mutation with the same expression profiles, and are therefore homogenous? If you have 100% transfection efficiency using a GFP reporter, for example, then at least you know that all of your cells were targeted for the mutation. Making mutations in mammalian cell lines is becoming much easier, especially with advanced molecular engineering techniques such as CRISPR/Cas9, among others. ![]()
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