Genomic Tail DNA Isolation From Transgenic Mice

(easy salt technique)

(of course, always follow the institutional care regulations. These are rough guidelines. Adapt as needed).

1. Using 3mm of tail tissue (hat's about half of a regular grain of white rice).

2. Cut tails  (0.5cm) and place in 1.5ml microfuge tubes, label with mouse id (scissors or blade, clean in between with 70% EtOH or 10% bleach)

3. Freezing is ok –20C (ok for 1 month or more)

4. To each tube, add 500ul ProtK Lysis Solution per tail (prep 495ul Lysis soln + 5ul PK per tail) ‡ 55C  from 4 hours to overnight (no need to shake)

5. Mix tube by inverting at the end of the digestion

6. Add 250 ul of Saturated NaCl, reclose tube

7. Cover the rack with another and invert 10X

8. Take tubes out of rack, place on ice 10 minutes

9. Spin tubes in a microfuge at 10K rpm for 5 minutes

10. With a p1000, transfer 750ul from the top of mixture to a clean, labeled tube with 1ml of 95% EtOH. Close the top, invert a few times to mix. (save the digested tail until you are finished, in case you lose the DNA pellet, you can go back and get more from the remainder)

11. A stringy white DNA precipiate should form. Spin it down 10K rpm, 60 sec

12. Decant supernatant and keep the DNA pellet

13. Rinse pellet with 70% EtOH (100ul) and invert over a paper towel to dry (15-30min)

14. Ressuspend in 120 ul TE for PCR. Can dissolve at 55C for 1 hour. Never vortex this genomic DNA! Always flick the tube with fingers to mix!

15. Use 1-2ul of this DNA for PCR. Use 1/10 to 1/5 for Southern Blot.


Solutions:

- Lysis Solution  (250ml, Room Temp)
Tris Hcl pH 7.4         17mM                    4.25ml      1M TrisHcl pH 7.4
EDTA            17mM                    8.5ml        0.5M EDTA
NaCl            170mM           8.5ml      5M NaCl
SDS             0.85%                           21.25ml    10% SDS


- Proteinase K Stock Solution (-20C):
20mg/ml in d2H2O

- MIX: 495ul Lysis  soln + 5ul PK Stock per tail



- Saturated NaCl:  To 50ml d2H2O in a conical tube, keep adding NaCl
and shaking, till it no longer goes into solution (precipitates at the
bottom). The aqueous phase is now saturated.