7.1. Genomic DNA Extraction

There are several protocols that can be used for DNA isolation. However, 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 a genetic screen or barcoded sample. 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 guides, hairpins, or barcodes present in the screened sample. As a result, the choice of DNA isolation protocol should be based on the number of cells in your samples.

We recommend the following protocols:

• For genomic DNA isolated from 10 million or more cells—which are typical from standard “dropout viability” CRISPR and RNAi screens with larger libraries—we highly recommend using the Large-Scale Genomic DNA Extraction Protocol. With this procedure, you usually obtain 50-100 µg of genomic DNA per 10 million cells. This amount of genomic DNA will overload most column-based DNA isolation kits and compromise yields. Conventional Genomic DNA Extraction is not column based and avoids DNA loss which can distort representation of the guides or barcodes in the population.

• 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 that contains 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.