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Transgenic & Gene-targeting Institutional Facility
Transgenic & Gene-targeting Institutional Facility  :  Protocol  :  DNA Fragment Preparation

DNA Fragment Preparation

  1. If requesting transgenics, purify the undigested plasmid containing the genes construct by either CsCl gradient or by a membrane column method (e.g., Qiagen Plasmid Maxi/Midi Kit or Promega Wizard Plasmid Purification Kit). We will need ~ 25.0ug DNA in TE buffer (10mM Tris, 1mM EDTA, pH 7.5; DNA concentration should range between 0.1 - 1.0ug/ul).
  2. The Transgenic Facility does the DNA fragment preparation ourselves. We ask that the vector be designed to be able to completely release and separate the transgene from vector/prokaryotic sequence.
  3. If requesting ES cell targeting, digest the vector with appropriate restriction enzyme(s) to linearize your construct (it is not necessary to remove vector/prokaryotic sequences).
  4. Isolate the linear DNA fragment by a phenol/chloroform extraction followed by a salt/ethanol precipitation.
  5. We need ~ 50.0ug of purified, linearized DNA in TE buffer (10mM Tris, 1mM EDTA, pH 7.5; DNA concentration should range between 0.5 - 1.0ug/ul).
  6. Bring the DNA fragment or plasmid, depending on the procedure, on wet ice to Lied B003.