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Section 3.2 Prepare a 2 × 25 bp mate-paired library Shear the DNA Table 32 3 Recommended shearing conditions for mate-paired library insert sizes Shearing method Shearing conditions 600 to 800 bp Covaris™ shearing in 20% glycerol • • • • • • • • • Number of Cycles: 75 Bath Temperature: 5 °C Bath Temperature Limit: 12 °C Mode: Frequency sweeping Water Quality Testing Function: Off Duty cycle: 2% Intensity: 7 Cycles/burst: 200 Time: 10 seconds 800 to 1000 bp Covaris™ shearing in 20% glycerol • • • • • • • • • Number of Cycles: 30 Bath Temperature: 5 °C Bath Temperature Limit: 12 °C Mode: Frequency sweeping Water Quality Testing Function: Off Duty cycle: 2% Intensity: 5 Cycles/burst: 200 Time: 10 seconds 1 to 2 kb HydroShear® Standard Shearing Assembly • SC5 ‡ • 20 cycles 2 to 3 kb HydroShear® Standard Shearing Assembly • SC9 • 20 cycles 3 to 4 kb HydroShear® Standard Shearing Assembly • SC13 • 20 cycles 4 to 5 kb HydroShear® Standard Shearing Assembly • SC15 • 5 cycles 5 to 6 kb HydroShear® Standard Shearing Assembly • SC16 • 25 cycles (13 mm × 65 mm borosilicate tube) (13 mm × 65 mm borosilicate tube) ‡ Speed code (SC): 5. IMPORTANT! If you are using the Covaris™ S2 System, set the chiller temperature to between 2 to 5 °C to ensure that the temperature reading in the water bath displays 5 °C. The circulated water chiller should be supplemented with 20% ethylene glycol. 2. If the DNA source is not limiting, ensure that the shearing conditions result in the desired insert sizes. Shear 5 μg DNA and run 150 ng sheared DNA on a 1% E-Gel® EX Gel according to the manufacturer's specifications. SOLiD™ 4 System Library Preparation Guide 95 Chapter 3 Mate-Paired Library Preparation Insert size
