- Each tail should be in a clean eppendorf tube.
- Add 500µl of tail lysis buffer containing Proteinase K (PK) to each tube.
- Incubate tail samples in 50-60C water bath overnight.
- Add 250µl saturated (6M) NaCl to each tube.
- Shake tubes vigorously (~ 20 times) and incubate tubes on ice for 10 minutes.
- Spin tubes on low speed (#6 on Hemle centrifuge) at 4C for 10 minutes.
- Remove supernatant and place into a clean eppendorf.
- Add 650µl isopropanol and invert to mix. Incubate tubes at room temperature for 15 minutes.
- Recover DNA by centrifuging, max speed, 10 minutes at room temp.
- Place tubes inverted on bench and allow to air dry 5 minutes.
- Add 200µl of TE pH 7.5 or sterile water to each tube. Incubate in 50-60C 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|
|0.5M EDTA pH 8.0||10mM||10ml|
Proteinase K concentration:
Add 20µl of a 20 mg/ml stock per 1ml of tail lysis buffer.
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 at 37C for 2 hours to overnight.
For important southerns:
- Dilute DNA in 400µl of water.
- P henol/chloroform extract DNA.
- Precipitate in 1/10 vol 3M NaOAc and equal volume of isopropanol.
- Precipitate 15 minutes at RT.
- Wash pellet with 70% EtoH.
- Resuspend in water.