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Screening Lambda gt11 with Antibodies 1. Plate lambda gt11 as described in "Plating Lambda" protocol. Use strain Y1090r-. Incubate plates
 

Summary: 99
Screening Lambda gt11 with Antibodies
1. Plate lambda gt11 as described in "Plating Lambda" protocol. Use strain Y1090r- . Incubate plates
for 3 hours at 42C 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 37C for 6 hours (4-8 hours is OK). Overnight (>8 hours) increases background.
4. Place plates at 4C 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 25C 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 25C 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).

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine