Centro de Astrobiologia (Instituto Nacional de Técnica Aeroespacial - Consejo Superior de Investigaciones Científicas)
Manufacture protocol
1. Acidiphilium sp. PM was grown aerobically at 30ºC in medium described in (San Martin-Uriz et al, 2013. Submitted). 2. A shotgun genomic DNA library of Acidiphilium sp. PM was constructed by digesting total DNA with Sau3AI. Fragments were separated by ultracentrifugation and those ranging from 2-6 kb, were ligated to BamHI-digested, Shrimp Alkaline Phosphatase-dephosporilated pBluescript II SK+ vector. Purified ligation products were then used to transform E. coli DH10B electrocompetent cells. 3. 8544 clones were picked, incubated overnight and lysed using alkaline lysis. After centrifugation, 2 µl of the plasmid-containing supernatant were used to PCR-amplify the inserts using the Expand Long Template PCR system (Roche). 84% of all PCR reactions were positive, with an average amplicon size of 2.2 kb. 4. PCR products were purified using 96-well plate DNA purification kits (TeleChem International, Inc., Sunnyvale, CA), then were spun-dried and resuspended in 1x MicroSpotting Solution Plus (Telechem International, Inc). 5. Purified PCR products were rearranged in 384-well plates with the aid of a liquid handling robot (epMotion 5075 Eppendorf, Hamburg, Germany). In this stage, controls were added. These include several Acidiphilium sp. PM genes involved in catabolism, replication, transcription and metal resistance. Several clones from a genomic library of Leishmania infantum were included as negative controls for the hybridization. 6. Finally, PCR products were printed in duplicate onto epoxy-coated slides (Telechem International, Inc.) at 21ºC and 37-44% relative humidity using a MicroGrid-TAS II Arrayer (Digilab, Inc., Holliston, MA).
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