Used to get long and high quality DNA for long-insertion DNA library preparation.
CTAB buffer (1x): preheat to 65°C
SDS-EB lysis buffer (can use the one inside the kit)
Proteinase K (20 mg/ml)
RNAase A (10 mg/ml)
M9 buffer
Wash the worms (starved, without bact) out of a 90 mm plate, using 3ml M9 and 2 1.5 ml tubes.
Collect the worm pellets from 2 tubes together by centrifugation at 3000 rpm.
Leave at most only ~50 ul M9 in the 1.5 ml tube.
Add 200 ul SDS-EB lysis buffer, and 25 ul Proteinase K, 55°C for at least 1h (up to 4h).
Mix well by inversion, every 5 min. (DONOT vortex: to avoid potential DNA shearing)
Add 250 ul pre-heat CTAB buffer to the tube, 65°C for 30 min.
Add 500 ul Phenol/chloroform (1:1) mixture to the tube, mix well by inversion (at least 1 min).
Centrifugation at 12000 rpm for 10 min, pipette the aqueous (about 450 ul) to a new 1.5 ml tube with an end-cutted tip.
Add 10 ul RNAase to the aqueous, RT for 30 min.
Add 1000 ul chloroform (1:1) to the tube, mix well by inversion (at least 1 min).
Centrifugation at 12000 rpm for 10 min, pipette the aqueous (about 400 ul) to a new 1.5 ml tube with an end-cutted tip.
Add 800 ul cold 100% ethanol to aqueous, leave sample at -80°C for at least 30 min.
Centrifugation at 7500 rpm for 10 min.
Wash the DNA pellet twice with 1 ml ethanol.
Discard the supernatant, air-dry the pellet (about 5-7 min is enough).
Resuspent the pellet in 200 ul TE buffer overnight at 4°C.