Extracting DNA from viruses embedded in agarose
Add agarose to SE buffer for a final concentration of 1.5% (wt/vol).
Melt the agarose in a microwave oven, then cool and maintain at 37°C in a water bath.
Warm the viral concentrate to 37°C in the water bath, then immediately mix with an equal volume of molten agarose and quickly transfer the mixture to casting molds.
Once the agarose has gelled, transfer the plug (or noodle from the syringe, or buttons from the parafilm), into a tube containing 5 volumes of lysis buffer amended with proteinase K (1 mg mL–1 final concentration).
Incubate at room temperature overnight.
Decant the lysis buffer, being careful not to lose the plugs.
Optional: Rinse the plugs twice, each time adding 25 volumes of fresh TE containing 1 mM PMSF, incubating for 1 h with gentle agitation, then decanting the rinse fluid.
Rinse the plugs, add 50 volumes of fresh TE with no PMSF, incubate for 30 min.
with gentle agitation, then decant the rinse fluid.
Once more, rinse the plugs, add 50 volumes of fresh TE with no PMSF, incubate for 30 min.
with gentle agitation, then decant the rinse fluid.
Store the plugs at 4°C submerged in TE.
