DNA Purification from Mouse Tail Clipping

DNA Purification from Mouse Tail Clipping

-cut tail (~.5cm) into 1.5ml Eppendorf tube

-add 500 ul of HMW cell lysing buffer (recipe to follow) and 10 ul of 10 mg/ml Proteinase K in water solution.

-incubate sample @ 50 degree from 6 hours to O/N. I always do overnight.

-spin down and take supernatant. (This step just clears out the dirt like hair, etc. You can skip this step with no problem if it saves time and tubes.)

Phenol-chloroform extraction:

-to supernatant, add 250 ul phenol and 250 ul chloroform (4ml isoamyl alcohol/96 ml chloroform) and mix very well.

-spin @ 14K rpm for 5 minutes.

-take top layer (w/o white precipitation) into new tube then add 500 ul 2-propanol and mix very well.

-spin sample @ 14K for 15 minutes at RT and discard supernatant using pipette.

-wash pellet with 300 ul 70% ETOH and spin for 5 minutes at RT. Remove ETOH and vacuum dry. Be sure your pellet is dry before continuing. You can let them dry at room temp as well if you prefer but it takes a lot longer.

Suspend pellet with:

- 100ul ddwater and RNase A mixture - final concentration 10 ug/ml (1 ul 10mg/ml RNase A in 1 ml of water).

-incubate at least 1 hour at 50 degree.

-do concentration measurement. This is optional once you have done it a few times just to see if your DNA is of good concentration. After a few litters, you may be able to drop this step. It’s up to you if you want to keep checking your DNA.