UTR200, Hielscher

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NB! pre-PCR operation

  1. prepare ice bath: ~2/3 of ice and ~1/3 of water;
  2. prepare DNA solution in degased 1x TE:
    0.5ml LoBindup to 5µg DNA in 50µl 1x TE
    2mlup to 20µg DNA in 400µl 1x TE

  3. insert plastic rod in the sample tube(s) [1];
  4. insert tubes tightly into floating holders; use tubes with water to fill empty holes;
  5. water/ice bath cooling for 5 min;
  6. run program at 100% amplitude (it is worth to check the water flow 2-3 times during shearing):
    fragment size [bp]cycletime [min]

  7. control shearing results by elecrophoresis[2];
  8. pool aliquots of sheared DNA, snap freeze in liquid nitrogen, store at -20°C;
  9. remove water from sonicator and tubings, dry sonicator with paper towel, relieve the tubing in peristaltic pump.

[править] Notes

  • sonicator does not require any special washing.
  • continuous water circulation is critical for the shearing, otherwise in ~10 min temperature becomes unacceptably high. It is necessary to control regularly (and change when necessary) the state of:
» peristaltic tubing (looses its elasticity and pumps very slowly)
» input filter and tissue prefilter
» needles

  1. use ~2.6cm long pieces of 1mm plastic rod to avoid liquid spraying to the walls and cap of a tube (in droplets on the walls and cap DNA would not shear efficiently); if you do not use plastic rod, it is worth to vortex/spin down sample tube(s) 2-3 times during shearing
  2. under-digestion happens regularly in some tubes for unknown reasons (normally, everything fine after repeat of digestion)

Личные инструменты


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