(last update : 14-09-1999)
Sucrose wash of cultured nematodes
Do this if the raw material (culture plate, soil extract, etc.) you want to extract gDNA from is rather messy and contains lots of bacteria, agar blobs, yeasts and other uninvited smudge. A sucrose wash will remove most of this smudge.
- Transfer the nematodes into a 15 ml centrifugable tube (e.g., if you start with a culture on one Petri plate, wash the worms off with 3 ml of the appropriate buffer and pipette them into a 15 ml tube).
- Rinse with 5 ml of an appropriate buffer (S-buffer for C. elegans and other insensitive beasts, P-buffer or plain water for wimpy worms of delicate complexion that hate salinity).
- Cool on ice for a few minutes.
- Spin 4 minutes at 3000 rpm and 4°C in a cooled centrifuge.
- Remove supernatant
.
- Add fresh buffer to a total volume of 4 ml.
- Add 8 ml 60% sucrose solution.
- Spin exactly 3 minutes at 3000 rpm and 4°C in a cooled centrifuge.
- Pipette or aspirate the floating froth (which contains the live worms) over to a new 15 ml tube.
- Rinse the walls of the old tube with buffer from a Pasteur pipette, to wash off worms sticking to the walls of that tube. Transfer the rinse to the new tube. BE CAREFUL to avoid mixing the buffer with the remaining sucrose in the tube.
- Add buffer to the new tube to a volume of 15 ml.
- Cool on ice for a few minutes.
- Spin 3 minutes at 3000 rpm and 4°C in a cooled centrifuge.
- Remove supernatant and you have clean worms.
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