Eccles:DNA Isolation

From OpenWetWare
Jump to navigationJump to search

Back to Eccles Lab > DGG Protocols

DNA Preparation.

I use a “homemade” version of the Promega Wizard genomic DNA isolation kit. Tail-tips can be frozen at -20C after collecting them (Up to 2 weeks is fine - probably can be left longer, but that's the longest I've left them).

  1. Add 600uL of nuclei lysis solution to each tail-tip in an eppendorf. To each tube add 10 uL of Protein Kinase (10mg/ml) and place on a shaker at 37C for 2-4 hours. If tissue has not properly dissolved by this stage, leave overnight at 4C (only hair and cartiledge should be present). If needed, you can leave the tail-tips at this stage o/n in the fridge.
  2. Add 200ul of 10M NH4OAc and vortex 20 seconds (each tube).
  3. Spin 5 minutes (13 000rpm). While spinning, set up new tubes with 600ul isopropanol.
  4. Add supernatants to isopropanol and invert slowly to see DNA spool into a visible mass. (You can leave them o/n in fridge at this stage if you want)
  5. Spin 5 min, carefully decant supernatant and air dry pellets (upside-down, 10-30 min at room temp). Wash pellet with 600ul room temp 70% ethanol. Spin again 5 min.
  6. Carefully decant supernatant and air dry pellets (upside-down, 10-30 min at room temp).
  7. Resuspend in 100ul Milli Q (or less if pellet is small- I usually use 50uL)

Some tissues (especially soft one like brain, liver etc or fetal tissue) it’s better and easier/quicker to homogenise the tissue in an eppendorf with one of those blue pestles with 600uL of nuclei lysis buffer, and then incubate at 65C for 15 minutes. Cool to room temperature, then add 200ul of 10M NH4OAc, and carry on with the rest of the protocol.


Nuclear Lysis Buffer

  1. 20mM Tris pH 7.5 - Use 4ml of 1M stock
  2. 50mM EDTA - Use 20 ml of 0.5M stock
  3. 1% SDS - Use 10 ml of 20% stock

Total volume 200ml

Trick to dissolving EDTA is to pH it to pH8, and keep stirring for a while, it does dissolve :-)