Sam:LabNotes/Microbiome-new/2010-9-9

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Revision as of 17:39, 10 September 2010 by >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…')
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DNA shearing test using Misonic sonicator 3000 - Pallson's lab

Background

  • MDA amplicons -> S1 nuclease treated -> mechanical sheared -> Purified -> 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.
  • 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