19 cDNA Library Screening
303
2. Incubation at 42°C is performed for 3-4 hrs (using E. coli Y1090) until
plaques become visible.
3. Plates are removed to a laminar flow hood where they are overlaid with a
nitrocellulose membrane that has been soaked in IPTG to induce expression from the phage lac Z gene. Foreach of the plates, a dry nitrocellulose
membrane is labeled with a soft pencil then soaked in the IPTG solution
in a petri dish. Excess fluid is at the rim of the dish drained off and the
membrane is then carefully lowered onto the bacteria lawn starting from
the center of the plate towards the rim. Filter membranes are always
handled with flat forceps.
4. Plates are placed inverted in an incubator overnight at 37°C.
5. Some plaques on the induction control plate will turn blue overnight, due
to expression of an intact ß-galactosidase gene, indicating successful
IPTG induction.
6. Before lifting the nitrocellulose membranes from the plates the position
of the membrane should be marked with three asymmetrically placed
needle sticks.
7. Nitrocellulose membranes are then carefully lifted off the plates and
placed contact side up on Whatman 3MM until all membranes have
been removed.
lmmunological staining
1. In order to wash off top-agar residue and cell debris a maximum of 5
nitrocellulose membranes at a time are transferred into a tray (10 x
10 cm) placed on a shaker and washed with 25 ml TBS for 2 x 10 minutes
at room temperature.
2. Non-specific binding sites on the filter membranes are then blocked by
incubating the filters for 30 minutes in 25 ml blocking buffer ( at least 5 ml
per one 90 mm membrane) at room temperature. This step is performed
with constant agitation to prevent filters from adhering to each other.
3. Each membrane is placed in a petri dish and individually incubated with
3 ml blocking buffer containing the preadsorbed primary antibody. A
typical working dilution of a polyclonal primary antibody is 1:100.
Such working dilutions of antibody can be reused up to 5 times and
are stored at 4°C. Incubation with primary antibody is performed at
Primary antibody
incubation
303
2. Incubation at 42°C is performed for 3-4 hrs (using E. coli Y1090) until
plaques become visible.
3. Plates are removed to a laminar flow hood where they are overlaid with a
nitrocellulose membrane that has been soaked in IPTG to induce expression from the phage lac Z gene. Foreach of the plates, a dry nitrocellulose
membrane is labeled with a soft pencil then soaked in the IPTG solution
in a petri dish. Excess fluid is at the rim of the dish drained off and the
membrane is then carefully lowered onto the bacteria lawn starting from
the center of the plate towards the rim. Filter membranes are always
handled with flat forceps.
4. Plates are placed inverted in an incubator overnight at 37°C.
5. Some plaques on the induction control plate will turn blue overnight, due
to expression of an intact ß-galactosidase gene, indicating successful
IPTG induction.
6. Before lifting the nitrocellulose membranes from the plates the position
of the membrane should be marked with three asymmetrically placed
needle sticks.
7. Nitrocellulose membranes are then carefully lifted off the plates and
placed contact side up on Whatman 3MM until all membranes have
been removed.
lmmunological staining
1. In order to wash off top-agar residue and cell debris a maximum of 5
nitrocellulose membranes at a time are transferred into a tray (10 x
10 cm) placed on a shaker and washed with 25 ml TBS for 2 x 10 minutes
at room temperature.
2. Non-specific binding sites on the filter membranes are then blocked by
incubating the filters for 30 minutes in 25 ml blocking buffer ( at least 5 ml
per one 90 mm membrane) at room temperature. This step is performed
with constant agitation to prevent filters from adhering to each other.
3. Each membrane is placed in a petri dish and individually incubated with
3 ml blocking buffer containing the preadsorbed primary antibody. A
typical working dilution of a polyclonal primary antibody is 1:100.
Such working dilutions of antibody can be reused up to 5 times and
are stored at 4°C. Incubation with primary antibody is performed at
Primary antibody
incubation