Protocols

DNA Fragment Preparation

  1. If requesting transgenics, purify the undigested plasmid containing the gene 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 25ug of DNA in TE buffer at a concentration of 1ug/ul.  (TE: 10mM Tris, 1mM EDTA, pH 7.5)
  2. The Transgenic Facility does the DNA fragment preparation.  Design your construct with unique restriction sites flanking the transgene, so the insert can be completely released from the vector/prokaryotic sequence.
  3. If requesting ES cell targeting, purify the undigested plasmid containing your targeting 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 100ug plasmid DNA in TE buffer at a concentration of 1ug/ul.
  4. Bring your plasmid DNA on wet ice to Hemenway G025.

Isolation of DNA from Mouse Tails

Day 1

  1. Cut 1cm of tail and place into a 1.5ml microcentrifuge tube, place on ice.  Add to the tube 0.62ul of Digestion Buffer (100mM NaCl, 50mM Tris-HCl, pH 8.0, 100mM EDTA, pH 8.0, 1% SDS) and 30ul of protease K solution (25mg/ml).
  2. Incubate at 55°C overnight on a rocking platform or in a water bath, mixing occasionally.

Day 2

  1. Cool the solution to room temperature.  Add 10ul of 1 mg/ml RNase A (DNase free) and incubate at 37°C for 1 hour, gently mixing intermittently.  Extract the aqueous supernatant once with buffered phenol (1 volume) and twice with phenol: chloroform: isoamyl alcohol (24:25:1 volume) shaking vigorously for 30 seconds and centrifuging at 12,000 rpm for 3 minutes for each extraction.  Remove final aqueous phase and place into a fresh microcentrifuge tube.
  2. Add an equal volume of isopropanol at room temperature and invert the tube several times until a precipitate forms.  Centrifuge 12,000 rpm for 30 seconds to pellet DNA, discard supernatant.
  3. Add 1ml of 70% ethanol (room temperature) to rinse pellet, centrifuge 12,000 rpm for 1 minute.  Discard ethanol and air-dry DNA pellet.
  4. Add 100-200ul of ddH2O or TE buffer (pH 8.0) to tube.  Incubate tubes at 55°C until DNA is dissolved.
  5. Measure the DNA concentration at 260 nm and 280 nm (OD260/280>1.75).  Use 2-10ug DNA for a Southern blot analysis or 100ng of DNA for a PCR reaction.

Last modified: Feb 09, 2012
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