Sam:LabNotes/Microbiome-new/2010-9-9
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MDA amplicons shearing using Misonic sonicator 3000 - Pallson's lab[edit]
Background[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 10uL sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)
File:Sam091010-sheared DNA.jpg
- 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.