Number of cells for starting the treatments

I have the following doubt. I've calculated that i need to get around 62 millions of cells after the puromicyn selection (i'm using the GecKo 2-vector library) to reach enough cell number to maintain the sgRNA representation of the library and also need to harvest around 37 millions to get enough gDNA to perform a 500X PCR for the NGS of this initial point. I plan to make 4 different conditions and here is where i have the doubts:

1) I have to start the 4 conditions with 62 millions cells as my initial number or i split them in four?

2) Is it better to infect more cells so after selection i can separate the 37 millions for gDNA extraction and use the rest (the 62 millions) for the different treatments?


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Fuchsia Kelpieover 4 years ago

Are the different conditions that you are testing different bioreps or different treatment conditions? If they are different bioreps, then you need 62 million cells each, if they are different treatment conditions, then you can split the initial 62 million cells into each treatment condition, provided that your cells grow at a reasonable rate such that after 1 week you will have 62 million in each treatment condition. Whenever I am setting up a screen for the first time, I always infect and maintain more cells than I need, just in case the cells do not necessarily grow as predicted, which happens often especially under antibiotic selection.

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