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='''MDA amplicons shearing using Misonic sonicator 3000 - Pallson's lab'''= ==Background== *MDA amplicons -> S1 nuclease treated -> mechanical sheared -> Size select -> Blunting(Polished) + DNA pol.I reparing -> Blunt ligation into vectors. *S1 nuclease digestion needs to be completely inactivated with the appearance of 0.5M EDTA, which can interfere the following enzyme reactions. *Using mechanical shearing save time on purification after S1 nuclease treatment. ==Plan== *Get access and training to use sonicator in Palsson's lab. *Test of shearing 1ug E.coli gDNA amplicons into 1~2kb for Sanger sequencing. Optimize the shearing time by aliquate sheared sample every 7 min. Validate sheared products on 2% E-gel. ==Procedure== *Sample preparation **Dilute 1ug 1ug of E.coli MDA amplicons into 100uL total volume with H2O. Transfer into a 1.5-mL microcentrifuge tube. **Prepare another tube with 100uL H2O for balancing. ===Operation manual=== *Hook up H20-tubes of cooling system. Release the white clipper. *Fill up H2O(tap H2O is acceptable) in the cup-horn to match the yellow tags *Turn on the switch of the cooling system. *Cooling system setting: Pump speed: "50", Desired temp "4C". *Turn on the switch of main controller. *Program setting: **Press "No" for not using micro-tip. **Press "program" to set up new conditions. **Tested condition: Pulse on(1min), Pulse(10sec), Output power(5), Temp monitoring(No). Total time(30min). *Press "start" to start the shearing. *Press "pause" to pause the shearing and aliqate sheared samples for validation. *After using, use the opposite order of operation procedures to turn off the machine. **Press "Stop" to stop shearing. **Turn off the main controller. **Turn down pump speed to "0" and turn off the switch. **For the cooling system, disconnect the H2O-tubes. Drain the H2O into the beaker. **Wipe out spilled H2O. *Technical support info: 800-323-4340 or 203-426-0101 ==Results== *10uL sheared DNA were validated using 2% agarose recycled E-gel (20uL/well) [[File:sam091010-sheared DNA.jpg|400px]] *It seems that even 7min (in the current pulse condition) is still too much for getting a 1~2Kb size fragments. **This result is reasonable because the Manual of Bioruptor sonicator(a very similar device)suggests to shear <5 min to get a 2~3Kb size (with 30sec on and off pulse). *I will try to optimize the shearing setting using new samples.
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