How to make HMW DNA agar plugs

Membrex as large a volume of water as feasible and spin the final retentate into pellets fo 15 min @ 12000 rpm

Take up the pellets in 1% SeaPlaque LMP agar. Keep the molten agar in a warm water bath to prevent it from solidifying. Do not use boiling agar. This will disrupt cells and their DNA.

Use 1-2 ml of 1% agar per pellet. Suspend the pellet well. You do not want chunks of cells in the agar. Transfer the agar to the plugmolds.

Let the agar solidify in the fridge for 30 minutes.

Break the end off one of the plug molds and push the plugs into lysis buffer :

Do not use more than 1 plug / ml of lysis buffer.

Incubate for 1-2 hours at 37˚C

Replace the lysis buffer with ESP buffer:

Incubate over night at 55˚C

Replace with fresh ESP buffer without Proteinase K

Incubate for 1 hour at 55˚C

Store the plugs in 0.5 M EDTA pH 8.0 in the fridge. Plugs should be stable this way for several years.