Screening Lambda gt11 with Antibodies
1. Plate lambda gt11 as described in "Plating Lambda" protocol. Use strain Y1090r- . Incubate plates
for 3 hours at 42°C until lawn and plaques are visible (2.5-4 hrs is OK).
2. Place on each plate a numbered nitrocellulose filter impregnated with IPTG. Place filter down on
plate carefully, starting at one edge. Once the filter touches the plate surface do not reposition it.
· Soak filters in 10 mM IPTG (~3 mg/ml; 2 ml/filter). Air dry on plastic wrap taped to bench.
3. Incubate at 37°C for 6 hours (4-8 hours is OK). Overnight (>8 hours) increases background.
4. Place plates at 4°C for 0.5 - 1 hour (optional). Reduces sticking of the filter to the top agarose.
5. Mark plate with ink-filled syringe and needle. Use asymmetric pattern of 3 - 4 holes. Wipe excess
ink off needle before use. Remove filters carefully.
6. Wash filters at 25°C in TBSTw (Tris buffered saline + 0.05% Tween 20) + 0.01% NaN3 + 0.5
mM EDTA. Do 1 quick wash, then 1 X 5 minute wash, followed by 1 X 10 minute wash.
· For washes, transfer filters one at a time to new container. Do this whenever washes are done
and/or buffers are changed. Use 5 - 10 mls per filter to ensure efficient washing. Swirl filters on
rotator set to slow/medium speed. Tween 20 (and Triton X-100 below) is present at 0.05%
(weight/volume). For the wash in step 6, add to TBSTw 1/1000 volume of 0.5 M EDTA, pH 9.
7. Block filters at 25°C in TBSTw + NaN3 + 10% heat inactivated fetal calf serum for 30 minutes.
8. Wash filters 3X in TBSTw: 1 quick wash; 1 X 5 minute wash; 1 X 10 minute wash.
9. Transfer filters to new container with primary antibody diluted in TBSTw + NaN3 (+/- EDTA).