Transgenic Mouse Core Facility
Mouse Tail Digestion Procedure
1. Prepare stock digestion buffer:
|100 ml||500 ml|
|5M NaCl||2.0 ml||10.0 ml|
|1M Tris-HCl, pH=7.5||5.0 ml||25.0 ml|
|0.5M EDTA||200ml||1.0 ml|
|10% SDS||5.0 ml||25.0 ml|
2. When ready to digest tails, aliquot the necessary amount of digestion buffer for the current set of tails (make sure the SDS is in solution! Heat it to 42C if necessary).
3. Add Proteinase K to a final concentration of 0.2mg/ml.
4. Add 0.5 ml of Proteinase K buffer to each tail in an eppendorf tube, vortex.
5. Incubate at 48-50C for several hours with occasional vortexing (this can go overnight).
6. After the tails have been completely digested (only hair and bones remaining), the samples can either be stored at –20C or immediately extracted with Phenol/Chloroform.
7. To begin extraction: Add 0.5ml of a 1:1 mix of Tris saturated phenol (with b-ME and hydroxyquinoline) and Chloroform (24:1 isoamyl alcohol). Vortex.
8. Microfuge at full speed for 3 minutes.
9. Remove aqueous phase to a new eppendorf.
10. Add 0.5 ml Chloroform (24:1 isoamyl alcohol). Vortex. Leave the aqueous phase on top of the Chloroform. The samples are now ready for PCR or they can be stored at 4C.
11. If doing a slot blot, aliquot 50ml of aqueous phase to a new eppendorf and precipitate with 125ml Ethanol.
12. If performing PCR, use 1-2ml of aqueous phase for a 50-100ml PCR reaction. Primers should be 50% GC, approximately 30 bases long, and used at a final concentration of 250mM. Our typical conditions are: 95C X 5’; 30(95C X 30"; 60C X 1.5’; 72C X 1.5’); 72C X 8’; 4C hold. All PCR reactions should be validated to detect a single copy transgene in the background of genomic DNA.