Plaque Lifts
1. For screening a library, plate approximately 1500-2000 plaques on each plate.
Incubate plates for 7-9 hours. Do not allow to get too large.
2. Store plates in fridge for 2 hours. Do not try to do lifts from warm plates, as the top
agar will stick to the filters.
3. Number the plates and a set of Hybond N or nitrocellulose filters. Wear gloves
when handling the filters.(Mark the filters using a pencil). Carefully lay the filters
onto the plate. DO NOT MOVE THE FILTER ONCE IT HAS MADE CONTACT
WITH THE AGAR Using a 26g needle that has been dipped in india ink, poke holes
through the filters into the agar. This will mark the filters and plates, so that the filters
can be correctly oriented with the plates after hybridization. Let the filters sit on the
plates for 2 minutes. If two lifts are to be made from a plate, keep the second filter on
the plate for 5 minutes. Do three or four plates at a time and keep the remaining plates
in the fridge, so they don't warm up.
4. Using forceps, carefully remove the filters and transfer them to Southern denaturing
solution for 1 minute, then to Southern neutralizing solution for 30 seconds, then to
2X SSPE (DO NOT USE SDS). When using nitrocellulose filters do not soak the
filters in the denaturing solution for longer than 2 minutes or they will get extremetly
brittle.
5. When all the lifts have been done, gently rub the filters to remove residual agar
(wear gloves). Drain the buffer and rinse the filters with fresh 2X SSPE.
6. Transfer the filters to 3MM paper to blot away remaining liquid. Air dry at room
temperature. Fix the DNA to the filters by UV irradiation, or by baking at 80oC for 2
hours. N.B. if nitrocellulose they must be baked under vacuum.