DIGESTION OF DNA IN AGAR PLUGS

Place no more than 5 plugs into 50 ml of 5% Sarcosyl and incubate at 37˚C for 2 hours.

Place plugs into new tube with 25 ml 20% Ethanol an incubate in the fridge for 2 hours on a shaker.

Equilibrate @ 4˚C for 1 hour.

Replace ethanol with TE and repeat equilibration.

Replace TE and repeat equilibration.

Replace TE with 1X of the appropriate digestion buffer without MgCl. For Hind III that is Promega buffer E (pH 7.5, 6 mM Tris, 100 mM NaCl, 1mM DTT).

Equilibrate @ 4˚C for 1 hour.

Replace the digestion buffer and repeat equilibration.

Place no more than 5 plugs into 1 ml of 5X digestion buffer.

Add 4 mM Spermidine and

Add 250 μl BSA

Add 200 U of Hind III / ml (1000 U total).

Bring up to 4.97 ml and diffuse the enzyme in over night at 4˚C.

Add 30 μl of 1 M MgCl (6mM final MgCl concentration).

Incubate plugs at 37˚C in floor-shake at 200 rpm for desired period of time.

To stop the reaction remove the plugs and place them into 0.5 M EDTA pH 8.0.