Quick and Dirty gDNA Prep (Crude gDNA extraction)

This method is an easy, quick and cheap way to prepare genomic DNA (gDNA) from a large number or clones. Note that the quality of this gDNA is poor compared to specialized gDNA extraction kits/methods.

Procedure:

1. Grow cells in a 96-well plate until confluent.

2. Aspirate media.

3. Optional: wash plate with PBS (aspirate PBS). Works just fine without, at least for 80/20 media.

4. Freeze plate at -80°C for at least 30 min.

5. Thaw plate at RT.

6. Resuspend cells with 50-100 µl/well TE buffer (pH 8.0) supplemented with 0.3 mg/ml proteinase K (Thermo Scientific Cat# EO0491). Triturate a few times to ensure complete lysis.

7. Transfer lysate to a PCR plate, seal the plate using an adhesive film, spin down briefly, and incubate at 50°C for 1 hr, 99°C for 10 min.

8. Cool down plate and briefly spin down.

9. The lysate is now ready to serve as a PCR template. Typical concentrations obtained from 80% confluent wells of mESCs are 100-300 ng/µl.

Note: when performing PCR screens on crude gDNA with GoTaq Green, increasing the annealing step length to 90 seconds helps (a lot!) PCRing accross long regions.