genomic DNA extraction problem

I’m preparing to extract genomic DNA for whole genome sequencing. We ever simply used the following steps and succeeded with acceptable DNA degradation extent.

  1. gather worms, 2. wash several times, 3. freeze at -80 degree, 4. thaw the worms and add proteinase K to 200~500 ng/ml, 60 degree digest 4 hours, 5. centrifuge and collect the supernatant to a new tube, 6. and phenol/chloroform to extract, 7. collect the supernatant, add 100% ethanol to precipitate gDNA, 8. 70% ethanol wash x2, 9. air dry and add ddH2O, 10. RNaseA treat.

However, recently the gDNA I got every time was degraded severely. I could get big DNA pellets after adding 100% ethanol and they were difficult to be dissolved completely (it’s also found in our successful cases) and the final concentration is fine when we used Nanodrop to measure it, however, gDNA on the gel is very weak with smear.

I tried to reduce proteinase K concentration and incubation time, use clean ddH2O, tubes, or use non-starving worms. But the problem is still not resolved.

Can anyone help?


Because of crosslinking in the cuticle, to properly digest worms you need to add a reducing agent such as beta mercaptoethanol to your proteinase step. Also, detergent.

I would also strongly recommend spooling your DNA instead of spinning it down, as this will remove (or at least greatly dilute) contaminants that are also insoluble in 70% ethanol but aren’t strongly associated with the DNA.

I’ve found the “preparing worm genomic DNA” section of Michael Koelle’s Southern Blot protocol to be very reliable (I’ve never done a Southern blot, I can’t speak to the rest of the protocol). I don’t think I found it necessary to use agarose plates as recommended in the protocol, and certainly didn’t bother with sucrose floatation.

Thank you so much! I’ll try this.

OR…DNEasy Blood and Tissue kit (Qiagen). Cuts out the uncertainties and the headaches. It’s a brave new world.

yaya, qiagen makes good stuff (duh) and we used the Gentra Puregene kit. It has a supplementary protocol optimized for worms.

I’ve always had good luck with Mike Nonet’s gDNA extraction. It’s worked well for me for long-range PCR to confirm MosSCI insertions.

I did Qiagen gentra Puregene protocol for WGS. Specifically, I used the “Gentra Puregene Tissue kit” (there are several protocols in the kit, and ittsue prep seemed to fit much better than cell culture preps) Worked just fine, but I didn’t do any comparisons.