How much DNA do I need to isolate prior to performing NGS?
How much DNA do I need to isolate prior to performing NGS?
Although you only need a small amount of DNA for sequencing, we recommend that you isolate DNA from 100–200 million cells for pooled screens. This typically amounts to 300–500 µg of genomic DNA. The reason for this is that it is essential to generate an NGS library that is representative of the diversity of the edited cells (and hence sgRNAs) still in the population after treatment. Purifying the DNA at this scale ensures that you can generate sequencing data for ~400–1,000 cells per individual sgRNA included in the library.
The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit includes the NucleoBond CB 500 kit for purification of genomic DNA. The NucleoBond CB 500 purification kit is provided in the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647) and contains columns and appropriate buffers to purify high-molecular-weight genomic DNA from 100–200 million cells from a mammalian cell culture. It is a maxi-size DNA purification kit that can purify up to 500 µg of very high-quality plasmid DNA.