6.1. Genomic DNA Extraction

To accurately measure the relative fraction of each sgRNA or barcode present in a specific cell population, it is important to isolate the whole amount of genomic DNA from the cells derived from genetic screen. Purification of genomic DNA from just a fraction of cells at a particular time point or treatment condition in a screen, may not provide full representation the effector constructs. There are several protocols that can be used for DNA isolation. Depending on the number and type of cells, some approaches may work better than others.

• Genomic DNA isolated from more than 10 million cells will overload most column-based DNA isolation kits and compromise yields. To avoid this loss of genomic DNA, which can distort representation of the guides or barcodes in the population, we highly recommend using the conventional Genomic DNA Extraction Protocol. With this procedure, you usually obtain 50 µg-100 µg of genomic DNA from 10 million cells.

This protocol is typically recommended to purify DNA from a “dropout viability” screen which often requires growth of 25-100M cells per time point or treatment variation, depending on the size of the library.

• For small and medium-sized populations of cells (from small libraries screens, positive selection screens where most of the cells are killed off, or FACS-based enrichment screens), we recommend using the following QIAGEN kits:
  • From 1 million to 10 million cells: Use the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Cat.# 69504)
  • For fewer than 1 million cells: Use the QIAGEN QIAamp DNA Micro Kit (QIAGEN, Cat.# 56304)
• After purification, you should resuspend your DNA at a concentration of ca. 1-2 µg/µl in water or Tris buffer (Important: Do not resuspend DNA in a buffer with more than 0.01M EDTA. It will inhibit the PCR reaction.). DNA samples can be stored at +4°C for a few weeks or at -20°C for an extended period of time.