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The two rounds of hybridization would generate a normalized population of tester specific cDNAs with different adaptors on each end.
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In this system, a population of bacterial tester cells carries a leaky lac frameshift mutation on a low-copy F′ lac plasmid.
The population of parental tester cells in the lawn of the selection plated was determined by taking three agar plugs from the surface of the selection plates, avoiding any Lac+ colonies.
sbs03: Subtractive library of a mixed stage population of clone 1B4 (tester) vs. mictic females with resting eggs of clone 1B4 (driver).
The y-axis show log transformed growth rates of tester populations.
(c) Growth of tester (indicator) populations.
Suppressive subtractive hybridization, or SSH [ 14], results in the isolation and amplification of mRNAs present in one population (the tester population) and absent in the other (the driver population).
sbs01: Subtractive library of a mixed stage population from a combination of cloned and non-cloned cultures (tester) vs. mixed stage population of a clone 1B4 (driver) sbs02: Subtractive library of a mixes stage population of clone 1B4 (tester) vs. a mixed stage population of a combination of cloned and non-cloned cultures (driver).
sbs04: Subtractive library of mictic females with resting eggs as tester vs. mixed stage population of clone 1B4 (driver).
This procedure allows for an enrichment of the population of cDNA that is specific to (differentially represented in) the respective tester cDNA sample.
To create the forward SSH cDNA library, which would represent enriched population of the overexpressed and newly induced transcript messages, TA1 and T2R were considered as 'Tester-A' and "Tester-B', respectively, and the C as the 'Driver'Driver
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