Worm Breeder's Gazette 11(4): 118

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Rapid Small Scale Worm Prep

Michael Granato, Heinke & Ralf Schnabel

Identification of a molecular polymorphism associated with a 
recombination event is a time consuming experiment because of the 
large number of DNA preps.  The following procedure has been used to 
isolate high molecular weight DNA which can be restricted from small 
quantities of worms.  The procedure avoids any CsCl centrifugation or 
the use of phenol and is therefore time saving.  The protocol makes it 
possible to process easily 20 samples within a day.  The yield is 
about 10  g from three 9cm plates.  
Procedure: Wash the worms ( starved L1 for highest yield) from the 
plates and purify from bacteria by standard sucrose flotation in a 1.
5ml cup.  Wash once with H2O and pellet the worms (about 0.2ml worm 
pellet).  Leave the cup at least 10 minutes on dry ice ( or store 
until use at -70 C) and add 0.5ml lysis buffer (50mM Tris-HCl,pH 8.5, 
100mM NaCl, 50mM EDTA, 1% SDS) and 30 l Proteinase K (10mg/ml) to each 
cup, invert gently until pellet is thawed and incubated in a 65 C 
waterbath for 2 hours.  Add 30 l Proteinase K, invert several times 
and incubated for another 2 h at 65 C.  Add 200 l 5M KAc to the 
viscous solution, invert gently and incubated for 30 minutes on ice.  
Centrifuge 3 minutes at 13k rpm on a bench centrifuge and transfer the 
supernatant into a fresh cup.  Add 2 volume of ethanol by overlaying 
the ethanol and mix by inverting the cup gently.  Leave 20 minutes at 
RT and wind up the precipitated DNA with a bent glass capillary.  
Transfer the DNA to a new cup with 200 l TE where it should dissolve 
easily.  Overlay with 200 l isopropanol and mix slowly by inverting 
the cup.  Leave 20 minutes at RT.  Again wind up the DNA as before and 
wash the DNA by placing it with the glass capillary in a cup with 400 
l 70% ethanol for 3 minutes.  Transfer the DNA as before into a cup 
with 40 l TE plus 100 g/ml RNase A.  Heat for 10 minutes at 65 C to 
dissolve the DNA and place the solution onto a millipore filter 
floating on TE in a 3cm petri dish.  Remove the dialysed DNA after one 
hour from the filter and check the concentration and quality on a 0.8% 
agarose gel.  Millipore filters: pore size 0.025 m (VSWP01300), place 
on TE, shiny side up.