Protocols

Numbering Mice and Isolation of Tail DNA

1. Using a 50 mL conical tube with a kimwipe saturated with isoflurane stuffed down inside it for each litter, place the 3 week old mouse inside until rapid breathing slows.
   NOTE: It is easy to kill the mouse if left exposed to the isoflurane too long!
2. When mouse is fully anethetized, punch ear (see core personnel for a guide or see Hogan's manual for key) and clip 1/2" off end of mouse tail. Store at -70°C until ready to prep.
3. Add 300 ul of tail buffer to eppendorf tube with tail.

   Tail Buffer: for 100 ml
   50 mM Tris (pH 8)
   5 ml of 1M stock
   
   100 mM NaEDTA
   20 ml of 0.5 M stock
   
   100 mM NaCl
   2.5 ml of 4 M stock
   
   1% SDS
   10 ml of 10% stock
   

4. Add 17.5 µl of proteinase K to each tube; vortex.
*proteinase K (10 mg/ml): dissolve 10 mg in 1 ml distilled H2O and aliquot in 200 µl into eppendorf tubes (freeze -20°C).
5. Incubate at 55°C overnight (tissue should dissolve leaving some fur).
6. Add equal volume (320 µl) phenol and vortex 10 seconds. Spin 15 minutes in microfuge at room temperature.
7. Remove aqueous top phase (300 µl) leaving interface behind and put into a clean eppendorf tube. Add an equal volume of phenol/chloroform and vortex 10 seconds. Spin 15 minutes in a microfuge at room temperature.
8. Remove aqueous top phase (300 µl) leaving interface behind and put into a clean eppendorf tube. Add an equal volume of phenol/chloroform and vortex 10 seconds. Spin 15 minutes in a microfuge at room temperature.
9. Remove aqueous top phase (300 µl) and put into a clean eppendorf tube. Add an equal volume of chloroform and vortex 10 seconds. Spin 15 minutes in a microfuge at room temperature.
   
10. Remove top aqueous phase (300 µl) and put into a clean eppendorf tube. Add 2 volumes (600 µl) 95% ethanol plus 50 µl 3 M NaOAC and mix (precipitate should form).
11. Use a 10 µl capillary tube to spool the DNA (DNA should readily stick to the glass capillary tube).
12. Insert DNA end of the tube into an eppendorf tube containing 100 µl 0.1X SSC and break off top of tube so the lid may close.
    (20X - SSC: for 100 ml - 17.5 g NaCl plus 8.8 g Na Citrate, q.s. dist. H20)
13. Spin EtOH tube in order to precipitate any remaining DNA for 15 - 20 minutes in refrigerated microfuge. Aspirate EtOH.
14. Resuspend DNA pellet in 20 µl of 0.1X SSC and transfer to tube containing 100 µl 0.1X SSC and spooled DNA.
15. Allow DNA to dissolve overnight. Read OD 260.
16. Store DNA at 4°C temporarily or at -20°C if long term storage is necessary.
    -OR-
    Complete steps 1 through 9 then:
17. Spin tube for 15 minutes. Carefully remove supernatant, leaving DNA pellet.
    
    
18. Add 600 µl 95 % EtOH and spin for another 15 minutes. Carefully remove supernatant, leaving DNA pellet.
19. Let air dry for 30 minutes.
20. Dissolve DNA in 75-100 µl of TE overnight at 37°C.