Extraction and PCR Analyis of DNA from Mice Tails

Place numbered Eppendorf tubes in dry ice.

Cut 0.5cm from end of tails and place in tubes.Store at -80℃ until analyzed.

Add 0.3ml of STE/PK solution to the Eppendorf tubes.

Incubate at 55℃ overnight with rocking or for several hours with occasional mild vortexing every 15min.

Add 2µl 10mg/ml RNase A solution.

Mix by mild vortexing and incubate 37℃ for 30min.

Cool to room temperature.Vortex 20s and spin for 5min.

Save supernatant （if pellet not visible，repeat previous step）and add 0.3ml isopropanol （2-propanol）.Mix by inverting tube 20 times.

Centrifuge for 5min.Discard supernatant.Rinse pellet with 0.3ml 70% EtOH.Spin briefly and pipette off remainder of EtOH.

Let air-dry for 30min，and add 50μl TE.Leave at room temperature overnight or heat for 1hour at 65℃ to dissolve.

Optional： measure OD₂₆₀ and use about 8µg DNA per lane if performing Southern analysis.

For PCR analysis，run thermocycler at （95℃x1min，63℃x1min，72℃x1min）x30； 72℃x5min； 4℃ end for，say，19-22-mer primer pairs that are less than 600 bases apart or （95℃x1min，72℃x3-4min）x40； 72℃x10min； 4℃ end for，say，48-mer primer pairs that are greater than 1500 bases apart.For the latter conditions，if DNA polymerase is added with a hot start，replenish the enzyme after about 1 hr.