For the initial set of experiments screening for the loss of a PstI restriction site situated between the PB1 recognition sites, the PCR amplified fragments were digested overnight at 37°C with 20 U PstI (New England BioLabs) in 1x NEbufferfer.
The deletion event is detected by screening the bacteria for a loss of tetracycline resistance.
The transfected cells were cultured in Zeocin containing medium and stable clones were screened for the loss or reduction of SKAP-55 greater than 90% and unaffectiveness of other proteins as described [8].
Next, erythromycin resistant clones were screened for the loss of the intron vector by replica plating, then erythromycin resistant and thiamphenicol sensitive clones were selected and further analyzed by PCR to determine whether the intron had integrated into its desired target site.
Colonies from the library were screened for the loss of arsenite oxidation or the loss of motility.
Several colonies representing each primary clone were picked and screened for the loss of the neomycin resistance cassette by PCR.
Clonal lines were screened for the loss of the neomycin-resistance cassette and for the absence of the Cre recombinase expression plasmid by PCR (Supplementary Table S1).
The resulting progeny is screened for the loss of white + and yellow + and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.
There is some loss of quality in images saved at 72 d.p.i., but on a computer screen, the loss is not very noticeable.
It is shown that for round wire mesh screens the loss coefficient is related to the porosity.
