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Worm DNA Isolation

Nematode Genomic DNA Isolation


Materials:

Rich Agarose Plates:

50 mM

NaCl

0.75 %

BactoPeptone

5 ug/ml

cholesterol*

1 mM

CaCl2*

1 mM

MgSO4*

25 mM

K-PO4 (pH 6.0)*

1.5 %

agarose (not agar)

* add after autoclaving

Worm Lysis Solution:

100 mM

Tris (pH 8.5)

100 mM

NaCl

50 mM

EDTA

1 %

SDS

1 %

beta-mercaptoethanol

100 ug/ml

proteinase K

Phenol / CHCl3

25:24:1

Phenol : CHCl3 : isoamyl alcohol

CHCl3

24:1

CHCl3 : isoamyl alcohol

Procedure:

  1. Grow up worms on three 150 mm Rich Agarose Plates until just before starvation
  2. Harvest worms
    • wash worms from plates with H2O and collect in 15 ml conical tube
    • pellet in centrifuge 30 seconds @ 300 - 500 x g
    • wash twice with H2O to remove bacteria
    • suspend worms in 1 ml H2O and transfer to 1.5 ml eppendorf tube
    • optional: can also wash worms by floating on 30% sucrose (final) and centrifuging 5 minutes
  3. Lysis of worms
    • pellet worms and suspend in 500 ul worm lysis solution
    • freeze worms at -80 degrees for >30 minutes
    • thaw tube and incubate 30-60 minutes @ 55-65 degrees C with occassional agitation
    • transfer solution to new tube leaving behind large debri (worm carcasses & egg shells)
  4. Clean up and precipitation of nucleic acids
    • extract solution (~500 ul) with equal volume phenol/CHCl3
    • extract aqueous phase with equal volume CHCl3
    • repeat extraction if necessary to further clean up aqueous phase
    • precipitate DNA by adding 2.5 volumes EtOH to final aqueous phase
    • pellet nucleic acids by centrifugation 10 minutes @ 16,000 x g
    • wash pellet 1-2 times with 70% EtOH
    • resuspend nucleic acids in 50 - 100 ul TE
  5. Optional: remove RNA by digestion with 20ug/ml RNaseA for 30 mintues @ 37 degrees

 

Miscellaneous:

References:

The Nematode Caenorhabditis elegans. editor: W.B. Wood (1988).

 

compiled by Chad Rappleye

Aroian Lab Protocols