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NB! pre-PCR operation

  1. prepare DNA sample:
      » concentration [µg/µl]:
      » volume [µl]:
      » amount [µg]:
    • estimate the shearing volume:
         --- shearing is performed in 125µl aliquots, up to 15µg DNA per aliquot ---
    • spin-filter DNA sample through 5µm filter:
         --- up to 700µl per one filtration; 5krpm, 1min (repeat, if sample does not come through) ---
    • wash filter 2 times with EB:
    • combine all flow-throughs ;
  2. enter program[1]:
    • shearing assembly: standard
    • number of cycles: 20
    • volume: 150µl
    fragment size [kb]speed code

    Open questions

    • Shearing for "1-2", "2-4,5", "2.2-5" were checked, and optimal speed code is not as in the manual. It is worth to check "3-6" and "4-8" digestions.
  3. shearing:
    • wash Hydroshear[2] before each sample (do not wash between aliquots of the same sample);
    • shear aliquote;
    • collect each aliquote in a new tube, keep in ice;
  4. wash HydroShear[2], store dry;
  5. analyse samples:
    • check size distribution on the gel for each aliquot,
    • repeat shearing "the whole program" for under-digested samples;
    • pool aliquots of the same sample
         --- to be sure, that all aliquots of the sample are the same ---
    • snap freeze in liquid nitrogen, store at -20°C;
  6. NH4Ac/EtOH precipitation;
  7. dissolve in total volume of EB:

[править] Maintenance

  • machine is extremely sensitive to the solid particles in the solution. If it clogs:
  1. remove sample from the machine;
  2. standard wash[2] (acid, alkaly, water; see below);
  3. change orientation of the orifice box, 2x was with water, restore orientation;
  4. try to shear sample further, if still cloged:
    • remove sample from the machine
    • standard wash[2]
    • change orifice box to a new
    • put old orifice box in water in 7ml tube (sonicate it later).
  5. disconnect the shearing assembly;

Sonication of the shearing module

  1. prepare sonication bath[3]:
    • bidistilled water till mark;
    • 50ml glass beaker on upside down "50ml tube holder"
  2. water wash:
    • put orifice boxes[4] in the beaker with bidistilled water[5]
    • put the beaker in sonicator, sonicate for 10 min.
  3. isopropanol wash:
    • put orifice box[4] in the beaker with isopropanol[5]
    • put the beaker in sonicator, sonicate for 10 min.
  4. repeat water wash;
  5. let the orifice dry on dust-free paper for 30 min. at room temperature;
  6. wrap into SaranWrap, store in polyethylene bag

[править] Notes

  • 5µm Filter is from Millipore (UFC30SV00)
  1. NB! the speed code necessary to obtain certain sizes may vary between shearing assemblies and also may change after sonication of an asembly
  2. 2,0 2,1 2,2 2,3 Washing Hydroshear
    • in the beginning and in the end of the work
    • 2x wash between different samples
    14x WS-I (0.2N HCl): single 2ml tubedo not submerge tubing too deep in the solution
    24x WS-II (0.2N NaOH): single 2ml tube
    34x WS-III (H2O):

            1st tube - 1st wash;
            2nd tube - 2nd, 3rd washes;
            2nd tube - 4th wash

    submerge tubing till the bottom of the tube to wash tubing outside

    * wash solutions (0.2N HCl, 0.2N NaOH, mQ) are 1.8ml aliquotes in 2ml tubes

  3. "Transsonic T420" / Elma, HF-frequency 35kHz
  4. 4,0 4,1 there should be no air bubbles in the orifice during sonication: remove with a pipette
  5. 5,0 5,1 sonicator’s water level should be ~1cm higher than the beaker’s water level
Источник — «http://www.molbiol.ru/wiki/Hydroshear»

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