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DNA Shearing

Ultrasound shearing:     UTR200     Covaris S2     Sonifier W-450D Hydrodynamic shearing:     Hydroshear     Nebulizer Notes  Comments

pre-PCR operation

Ultrasound digestion: UTR200 / Hielscher

1. prepare ice bath: ~2/3 of ice & ~1/3 of water;
2. prepare DNA solution in degased 1x TE:

   tube aliquotes 2ml up to 20µg DNA in 400µl 1x TE 0.5ml LoBind up to 5µg DNA in 50µl 1x TE

3. insert tubes tightly into floating holders; fill empty holes with tubes with water;
4. start water/ice bath cooling;
5. run program at 100% amplitude (it is worth to vortex/spin down tubes & check the water flow several 2-3 times during shearing):

   Fragment Size [bp] Cycle Time [min] 100-200 50% 120 200-250 60 250-300 30 300-400 25% 30 400-500 20 500-600 10 600-800 5

NB! it is necessary to control shearing results by elecrophoresis, because under-digestion happens regularly in some tubes for unknown reasons
6. pool aliquots of sheared DNA into 1,7ml (labelled #lib) tube, snap freeze in liquid nitrogen, store at -20°C;
7. remove water from sonicator and all tubings, dry sonicator with paper towel, relieve the tubing in peristaltic pump

• detergents (SDS) suppress shearing;
• do not influence digestion:
  • glycerol up to 50%;
  • betaine up to 3M;
  • 2µm glass beads.

Maintenance

• sonicator does not require any special washing;
• continuous water circulation is critical for the machine, because otherwise in ~10min temperature becomes unacceptably high. It is necessary to control regularly (and change when necessary) the state of:
  • peristaltic tube;
  • input filter and tissue prefilter;
  • needle.

Ultrasound digestion: Covaris S2

will be added

• less convenient than UTR200;
• glass tube is for better heat dissipation.

Maintenance

• daily (when in use): change bidestilled water;



• monthly:
  1. turn off degasser;
  2. empty and wipe clean acrylic tank;
  3. wipe clean all accessible parts;
  4. fill tank with 1% bleach (1/l0 dilution of stock solution Na hypochlorite);
  5. turn on degasing for 10min;
  6. repeat three times:
       ◊  ◊  ◊   empty, rinse & fill acrylic tank with normal water;
       ◊  ◊  ◊   degas for 30 sec;
  7. turn on degasing for 10min;
  8. repeat three times:
       ◊  ◊  ◊   empty, rinse & fill acrylic tank with bidistilled water;
       ◊  ◊  ◊   degas for 5 min;



• keep tube holders for pre- and post-PCR applications separately;

Ultrasound digestion: Sonifier W-450D / Branson

1. dilute 1ng-1µg of DNA in 0.4-1ml of degased 1x TE;
2. put DNA in 2ml non-autoclaved tube, cool it on ice;
3. bring to the sonifier:
   
   • 1L mQ water,
   • 700ml glass beaker,
   • yellow tips,
   • parafilm pieces,
   • paper towels and lens cleaning tissue,
   • ice
4. wash the sonotrode two times in ~300ml mQ (40% amplitude; 10sec);
5. dry the sonotrode (40% amplitude; 10sec), remove the rest of water with lens cleaning tissue;
6. open the tube with DNA, cover it with parafilm and make a hole with a yellow tip;
7. insert the sonotrode to about 1/2 level of the solution / make sure the sonotrode does not touch the walls;
8. plunge the tube into the beaker with water/ice;
9. 10 min of sonication (10% amplitude; sonication/cooling = 10sec/50sec; program 6; total time is ~1h);
10. remove the tube with DNA, snap freeze in liquid nitrogen, store at -20°C;
11. wash the sonotrode again.

Maintenance

Wash sonotrode in Na hypochlorite between DNA samples:
• incubate 30min in 1% Na lhypochorite;
• wash thorougthly in water.

Hydroshear

1. spin-filter DNA sample through 5µm filter;
2. aliquote in 1,7ml tube up to 15µg DNA;
3. adjust volume of each aliquot to 125µl with EB;
4. wash Hydroshear;
5. run program (do not wash Hydroshear between aliquots of the same sample):
   • shearing assembly: standard;
   • number of cycles: 20;
   • volume: 150µl;

   fragment size program 2-4,5kb SC10 2,2-5kb SC12 3-6kb SC14 4-8 kb SC16

6. sample:
   • collect in a new tube;
   • keep in ice, check size distribution on the gel;
   • snap freeze in liquid nitrogen;
   • store at -20°C;
7. wash HydroShear after the sample, store dry;

Maintenance

• wash (i) in the beginning, (ii) after the end of the work and (iii) between samples:

  stage operation comments 1 4x WS-I (0.2N HCl): single 2ml tube do not submerge tubing too deep in the solution 2 4x WS-II (0.2N NaOH): single 2ml tube 3 4x 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




• machine is extremely sensitive to the solid particles in the solution. If it clogs, there is a chance to wash it, temporallry changing the orientation of the shearing module. Another optionis is to sonicate the shearing module.
• wash solutions (0.2N HCl, 0.2N NaOH, mQ) are 1.8ml aliquotes in 2ml tubes.

Nebulizer

1. attach vinyl tube to the nebulizer;
2. dilute 1ng-10µg of genomic DNA in 50µl of TE;
3. add Nebulization Buffer up to 700µl of, mix & put in the nebulizer;
4. chill nebulizer in ice: ~10 min;
5. connect air through the 0,2µm filter unit;
work under fume hood!
6. shear DNA (in ice) for 6min at 32-35psi (2,2-2,4 bar);
7. collect DNA by 1 min. centrifugation at 4°C ~500g (1400rpm Beckman J6-HC) / should be ~400-600µl;
8. snap freeze in liquid nitrogen, store at -20°C;



Nebulization buffer: 50% Glycerol in TE (ρ~1.1g/ml);

• mean size of the fragments is ~300-400bp;
• ~30-50% of sample volume is dissapeared as a fog;
• it is unpractical to increase shearing time, it does not decrease the size of fragments, but more liquid will be lost as a fog;

Maintenance

In principle, it should be possible to reuse nebulizer for some applications. Wash it by several cycles of nebulizing pure water.

Notes

UTR200 Covaris S2 Sonifier W-450D Hydroshear Nebulizer shearing force ultra sound hydrodinamic shearing is not effective for DNA fragments <0.3kb <1kb shearing size 0.1-1.5kb 1.5-8kb 0.2-1.5kb digestion time per sample ~2h per 6 samples ~20 min ~30 min ~20 min ~20 min contamination sensitivity closed tube wash between use wash between use single use price very low ~???