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 .
