Waldminghaus:Back Door/Sonication

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(New page: =Sonication= Decide wether you would like to sonicate in a 15 ml Falcon tubes or in a 1,5 ml Eppendorf tube. In a 15 ml Falcon tube you can only fill in a maximal volume of 1,7 ml. In ...)
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Revision as of 13:08, 3 November 2012

Sonication

Decide wether you would like to sonicate in a 15 ml Falcon tubes or in a 1,5 ml Eppendorf tube.

In a 15 ml Falcon tube you can only fill in a maximal volume of 1,7 ml. In an 1,5 ml Eppendorf tube you can only fill in a maximal volume of 300 ul (otherwise the sonication will not work out very well).


Procedure

  • Resuspend your bacteria pellet in IP buffer.
  • Sonicate for 30 s at high level i.e. 48 times in Eppendorf tubes if you wish to have chromosomal E. coli DNA at a size of around 200 – 500 bp.
  • Cool the samples for 30 s between the 48 cycles.
  • After sonication centrifuge for 10 minutes, 4°C at around 7150 g.
  • Take the supernatant (= sheared DNA) and then go on with your further procedure.
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