Another Bluescript anecdote:
I, too, have purified lovely, lit Lambda-ZAP plaques to homogeneity
only to find that only vector reacts with the probe. I'm still not too
clear why only certain lambda acted this way, but...
The two successful solutions:
1. Antibody screens
2. "R408 helper phage was first used to rescue and package pools of
single-stranded phagemid DNA from aliquots of the lambda ZAP library
containing approximately 10 to the 7 lambda phage. The procedure
paralleled the Stratagene protocol for rescue of plasmids from individual
lambda ZAP clones, and yielded about 5 ml of phagemid stock. After
incubation of 200 ul of phagemid stock with 200 ul of E. coli XL1-Blue
(A550 = 1) for 20 min at 37 degrees C, aliquots containing about 4000
ampicillin-resistant colony-forming units were used to inoculate cultures
for DNA minipreps. The resulting pools of plasmid DNA were cut with
EcoRI to release inserts, fractionated by agarose gel electrophoresis,and
transferred to nitrocellulose.... " * If a pool contained an insert that
hybridized with my probe, I went back to the miniprep that was the
source of that plasmid pool. I was able to use colony lifts at this point,
since my problem was more with Lambda than the Bluescript phagemid,
but there's no reason one couldn't use the gel technique to narrow things
down further and further....and eventually to the clone with your favorite
By the way, the numbers above were derived AFTER I verified an
antibody positive plaque by sequencing. I spiked with different
concentrations of my positive to see what kind of pools I could use and
still see the thing well.
*Lifted from K.M. Schmid, J.B. Ohlrogge. 1990. A root acyl carrier
protein-II from spinach is also expressed in leaves and seeds. Plant
Molec. Biol. 15: 765-778.
Dept. of Biological Sciences
4600 Sunset Ave.
Indianapolis, IN 46208
schmid at butleru.edu