DNA Fragment Preparation
- 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)
- 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.
- 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.
- Bring your plasmid DNA on wet ice to Hemenway G025.
Isolation of DNA from Mouse Tails
- 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).
- Incubate at 55°C overnight on a rocking platform or in a water bath, mixing occasionally.
- 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.
- 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.
- Add 1ml of 70% ethanol (room temperature) to rinse pellet, centrifuge 12,000 rpm for 1 minute. Discard ethanol and air-dry DNA pellet.
- Add 100-200ul of ddH2O or TE buffer (pH 8.0) to tube. Incubate tubes at 55°C until DNA is dissolved.
- 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.