1、Put a 0.5-1.5 CM MOUSE TAIL SNIP in a 1.5 ml eppindorf tube and store frozen until digestion.Make certain that the eppindorf tubes seal well,otherwise your samples may leak out during the shaking steps (below).

2、Add 600 ul TNES buffer (Warm the buffer until it is in solution before using);can leave tail whole or cut into pieces.(We leave it whole.)

3、Add 350 ug of proteinase K.(17.5 ul of a 20 ug/ul stock.)

Note:Since we do many tails at a time,we combine steps 2 and 3.That is,we make a master mix of TNES and proteinase K and aliquot 617.5 ul of the mix into each tube.Leave the master mix at room temp;do not put it on ice as this will cause the TNES to precipitate.)

4、Incubate at 55°C,200 rpm for 1 hour or UNTIL TISSUE IS COMPLETELY DIGESTED (ONLY HAIR WILL REMAIN).

Note:Shaking the samples will speed up the digestion process,but it is not absolutely necessary.Also,the amount of time required to digest the tails will vary depending on the size of the tails.It is imperative that the tails are completely digested before you proceed with the steps below.

5、Add 167 ul saturated (~6M) NaCl.Shake vigorously or vortex for 15 sec.

6、Centrifuge at 14000 rpm for 10-15 min.

Note:The original protocol calls for a 10-15 minute spin.I generally spin for 20-30 minutes.Either way will work, but I find that a 20-30 min.spin makes the next step easier.

7、Remove supernatant and put in a clean 1.5 ml eppindorf tube.

Note:When removing the supernatant you want to minimize carry over of the pelleted debris.If you transfer too much debris into the clean tube,simply centrifuge this tube and transfer the supernatant into a fresh tube.

8、Add an equal volume of cold,95% ethanol.Gently invert tube several times to precipitate DNA.

9、Centrifuge 20-30 minutes at 14000 rpm,4°C.

10、Drain off alcohol.Wash pellet with cold 70% ethanol.(70% ethanol wash is optional.)

11、REMOVE ALL RESIDUAL ALCOHOL AND DRY THE PELLET.DO NOT OVERDRY!Resuspend pellet in 200 ul water (for 0.5 cm tail snips) or 400 ul water (for 1.0-1.5 cm tail snips).  Use 1 ul for PCR.  Centrifuge the sample at high speed for 2 minutes before removing an aliquot for PCR.  Also, pipet from the top of the sample to avoid any debris that has settled at the bottom of the tube.

                                                                                          For 100 mls:TNES buffer (pH 7.5):                   50 mM Tris                           5 ml 1M Tris (pH 7.5)                                                  0.4 M NaCl                           8 ml 5M NaCl                                                 100 mM EDTA                      20 ml 0.5 M EDTA                                                 0.5 % SDS                            5 ml 10% SDS                                                                                             up to 100 ml w/sddH2O