C. elegan DNA extraction

Model Organism: Worms Scientific Discussion
1

The following is a DNA extraction protocol that i have found online. Does anyone know the ratio of phenol/chloroform that should be used?

C. ELEGANS GENOMIC DNA PREPARATION
Materials and Reagents:
Plates: NGM plates overlaid with 1% agarose—agar contains material that can inhibit
subsequent enzymatic manipulations of DNA
Lysis Buffer: 200mM NaCl, 100mM Tris-HCl pH 8.5, 50mM EDTA, 0.5% SDS
-aliquots are usually kept at -20°C
-add Proteinase K to 0.1mg/mL before use
phenol/chloroform
3M Sodium acetate
100% ethanol
70% ethanol
Procedure:

  1. Wash worms from plate (NGM-AGAROSE) in dH2O;
    this can be done in any size tube, but it is convenient to split worms so that
    subsequent steps can be done in eppendorf tubes.
  2. Pellet by spinning at 3000rpm for 2min.
  3. Remove supernatant and add 5 volumes of lysis buffer with Proteinase K
    (~100mL pellet of worms is convenient).
  4. Incubate at 65°C for 1-2hrs.
  5. Incubate at 95°C for 20-30min.
  6. Add DNAse-free RNAse to 0.1mg/mL and incubate at 37°C 1 hour.
  7. Extract 3 times with 1 volume phenol/chloroform.
  8. Add 0.1 volume 3M sodium acetate and >2 volumes 100% ethanol.
  9. Mix and incubate at RT or in the freezer for at least 1hr.
  10. Pellet DNA by centrifugation at 14000rpm for 15min.
  11. Remove supernatant and wash pellet with 70% ethanol.
  12. Air dry and resuspend in dH2O or TE.
    Tips/Troubleshooting:
  13. 3X phenol extraction should be sufficient, but if white precipitated material is
    still visible at aqueous/organic interface, repeat extractions until it is no longer
    visible.
  14. This prep is typically clean enough for PCR or restriction digests. If DNA is
    to be used for injection into animals, you may wish to clean up DNA over a
    spin column after restriction digest.
  15. Some protocols call for a freeze/thaw step to be included before lysis, but this
    does not seem to be necessary.
    Reference: Modified from Silverman lab protocol
    http://web1.tch.harvard.edu/silverman/protocols/
    Submitted by: James Morley 5/03

Thank you,
Ryan Puccia
Culver Academies
High School Senior

2

It is not actually necessary to clean up the worm lysate after running it through the thermocycler. If you still want to for the sake of purity, then a 50/50 mixture of phenol/chloroform is used, and for best results it should be saturated with a buffer such as TB (you can buy saturated phenol/chloroform premixed.

3

Do you want to extract DNA for a PCR?
If yes you can also just put 3-5 worms into a PCR tube and proceed to your PCR reaction.
Use a heat activated Taq! During the 10 minute heat activation the worm will lyse, also the antibodies coating the inactivated Taq will protect it from proteases released from the boiling worm.
The DNA released from these worms will be enough to PCR amplify your genomic region of interest.

Good luck,
Marlon