Sam:LabNotes/Microbiome-new/2010-9-9: Difference between revisions

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>Sam Chiang
(Created page with '='''DNA shearing test using Misonic sonicator 3000 - Pallson's lab'''= ==Background== *MDA amplicons -> S1 nuclease treated -> mechanical sheared -> Purified -> Blunting(Polishe…')
 
>Sam Chiang
 
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='''DNA shearing test using Misonic sonicator 3000 - Pallson's lab'''=
='''MDA amplicons shearing using Misonic sonicator 3000 - Pallson's lab'''=


==Background==
==Background==
*MDA amplicons -> S1 nuclease treated -> mechanical sheared -> Purified -> Blunting(Polished) + DNA pol.I reparing -> Blunt ligation into vectors.
*MDA amplicons -> S1 nuclease treated -> mechanical sheared -> Size select -> Blunting(Polished) + DNA pol.I reparing -> Blunt ligation into vectors.
*S1 nuclease need to be completely inactivated with the appearance of 0.5M EDTA, which can interfere following enzyme reactions.
*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.
*Using mechanical shearing save time on purification after S1 nuclease treatment.


Line 16: Line 16:


===Operation manual===
===Operation manual===
*Hook up H20-tubes
*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.

Latest revision as of 17:51, 16 January 2011

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.