Jacks Lab,Center for cancer research,MIT

1.Each tail should be in a clean eppendorf tube.

2.Add 500µl of tail lysis buffer containing Proteinase K (PK)to each tube.

3.Incubate tail samples in 5060℃water bath overnight.

4.Add 250µl saturated (6M)NaCl to each tube.

5.Shake tubes vigorously (~ 20 times)and incubate tubes on ice for 10 minutes.

6.Spin tubes on low speed (#6 on Hemle centrifuge)at4℃for 10 minutes.

7.Remove supernatant and place into a clean eppendorf.

8.Add 650µl isopropanol and invert to mix.Incubate tubes at room temperature for 15 minutes.

9.recover DNA by centrifuging,max speed,10 minutes at room temp.

10.Place tubes inverted on bench and allow to air dry 5 minutes.

11.Add 200µl of TE pH 7.5 or sterile water to each tube.Incubate in 5060℃water bath for * 10 minutes.Resuspend pellet by pipetting up and down several times.

Tail Lysis Buffer:

Final Concentration per 500ml

1M Tris pH 8.0  10mM 5ml

5M NaCl  100mM 10ml

0.5M EDTA pH 8.0 10mM 10ml

10% SDS  0.5% 25ml

dH2 0  to 500ml

Proteinase K concentration:

Add 20µl of a 20 mg/ml stock per 1ml of tail lysis buffer.

ES Cells:

For ES Cells the protocol is very much the same except for the following:

All steps are done in a well of a 24 or 6-well dish.

The initial incubation in the lysis buffer is done at37℃for 2 hours to overnight.

Southerns:

For important southerns:

1.Dilute DNA in 400µl of water.

2.P henol/chloroform extract DNA .

3.Precipitate in 1/10 vol 3M NaOAc and equal volume of isopropanol.

4.Precipitate 15 minutes at RT.

5.Wash pellet with 70% EtoH.

6.Resuspend in water.