Shreffler:DNA Isolation for JAX

Materials

• RBC Lysis Solution (Qiagen 158904)
• Cell Lysis Solution (Qiagen 158908)
• 50 ml conical tube

Procedure

1. Add 5 ml blood to 15 ml RBC Lysis Solution. Invert to mix, and incubate 20 min at room temperature on a nutating mixer/rocker.
2. Centrifuge for 5 min at 2,000 x g. Carefully aspirate supernatant, leaving behind the visibile white cell pellet and about 200-400 µl of residual liquid (cell pellet may still be red at this point). If you are aspirating from multiple samples, use a different pasteur pipet for each sample as to not cross-contaminate.
3. Vortex tube vigorously to resuspend cells in residual liquid.
4. Add 10 ml RBC Lysis Solution. Invert to mix, and incubate for 5-10 min at room temperature on nutating mixer.
5. Centrifuge for 5 min at 2,000 x g. Carefully aspirate supernatant, leaving behind the visible white cell pellet and about 200-400 µl of residual liquid.
6. Vortex tube vigorously to resuspend cells in residual liquid.
7. Add 5 ml Cell Lysis Solution. Vortex vigorously for 10 seconds to lyse the cells, but take care to start from slow speed and gradually move to higher speed, helping to minimize foam buildup.
8. Incubate overnight at room temperature on nutating mixer to ensure that samples are homogeneous (no cell clumps).
9. Parafilm seal the cap of the tube and ship to Shrefflab for storage. (if storing for a couple days prior to shipping, store at 4°C)