title: LBEG Dmel/UChile
status: Public on Jul 01 2009
contact.city: Santiago
contact.first: Alejandro
contact.last: Zúñiga
contact.email: jano@inta.cl
contact.address: Macul 5540
contact.country: Chile
contact.institute: INTA - Universidad de Chile
organism: Drosophila melanogaster
manufacture_protocol: Clones were picked from -80°C stocks and grown overnight in 96 well plates in 200 µl LB plus 50 µg/mL of ampicillin. PCR amplification of bacterial cultures and quality verification of the products were performed as described in González-Agüero et al (2008). PicoGreen ds-DNA Quantitation Kit (Molecular Probes) was used according to manufacturer instructions to quantify and standardize PCR products concentration. Twenty µl of the PCR products were arrayed in 96-well plates and mixed with an equal volume of 50% dimethyl sulfoxide. PCR products (70 ng) were spotted in duplicate on 8 x 12 cm Nylon membranes (GeneScreen Plus, Biorad) using an 8 pins print-head (Arraylt model SSP015) and the arraying robot Versarray Chip Writer Compact (BioRad). The membranes were treated as described in González-Agüero et al. (2008). In addition to the library clones, the following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T7 and SP6 universal primers; (2) several spots of DMSO 50%; (3) PCR amplified fragment of genes serendipity α, twist, tinman, fog and snail as positive controls, and (4) actin, tubulin, and RP49 as housekeeping, and (5) four dilutions of a PCR amplified fragment from a B. subtilis dap cDNA (ATCC; number 87486), which hybridizes to an in vitro synthesized poly(A)-RNA that was added to the embryo RNA samples (dilution 1/200) prior to the labelling process and used as spike mRNA (Kane et al., 2000).