Wood Book Small Scale DNA Prep ver 14 sep 1994

Essentially from the Methods Section of the book The nematode Caenorhabditis elegans, Cold Spring Harbor Press, WB Wood ed. (J. Sulston and J. Hodgkin)

WLB2 is  final                                       to make 10 ml
                110 mM NaCl                        1.1 ml of 1 M
                110 mM Tris-Cl, pH 8.5       1.1 ml of 1 M
                55 mM EDTA                        2.75 ml of 200 mM
                1.1% SDS                             1.1 ml of 10%
                1.1% 2ME                             110 µl of 100%
                                                              3.84 ml H2O
Use 450 µl and add 50 µl proteinase K at 1 mg/ml

1 WORM PREPARATION
Wash three 9 cm plates-worth of worms thoroughly in M9 buffer
Float over 1 M sucrose (this removes agar components which inhibit enzymes)
Wash twice with M9
Pellet in a 1.5 ml tube (making a 100 µl pellet)
Freeze at -70°C

2 LYSIS AND DIGESTION
Thaw and add immediately 0.5 ml of WLB2:
Incubate 30 min at 65°C with occasional very slow inversion

3 PHENOL EXTRACTION and PRECIPITATION
Extract with 0.5 ml phenol:
-add organic reagent
-mix by very slow inversion for 5 min or more
-spin 10 min at 12000 rpm
-remove lower organic phase
Extract with 0.5 ml phenol/chloroform (id.)
Extract with 0.5 ml chloroform (id. - BUT transfer supernatant with wide tip to new tube!)
Add 1 ml 100% ethanol (kept at -20°C) to final 0.4 ml supernatant
Spin 15 min at 12000 rpm
Wash pellet twice with 70% ethanol:
-remove ethanol
-add 0.5 ml 70% ethanol, mix and spin
Wash pellet once with 95% ethanol
Extra spin to remove last ethanol
Resuspend in 50 µl TE (takes > 1 hr)