title: Complete shotgun genomic Leishmania infantum DNA microarrays
status: Public on May 14 2009
contact.city: MADRID
contact.first: PEDRO
contact.middle: JOSÉ
contact.last: ALCOLEA ALCOLEA
contact.email: pjalcolea@cib.csic.es
contact.address: Ramiro de Maeztu, 9
contact.department: CENTRO DE INVESTIGACIONES BIOLÓGICAS
contact.country: Spain
contact.institute: CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS
organism: Leishmania infantum
description: epoxy
manufacture_protocol: Leishmania parasites were grown in RPMI 1640 supplemented with glutamine, 10% heat inactivated foetal bovine serum and antibiotics. A complete shotgun genomic library of Leishmania infantum (32,6Mbp) was constructed by digesting genomic DNA with Sau3AI restriction enzyme. After digestion, fragments ranging from 2 to 10 Kb were separated by ultracentrifugation through a 10-40% sucrose gradient. pUC18 vector was digested with BamHI, thus yielding compatible blunt-ended fragments, that were ligated with the purified set of inserts with T4-DNA ligase. Finally, DH10B electrocompetent E. coli cells were transformed. The efficiency of this L. infantum shotgun DNA library was 95%.We picked 29952 colonies, incubated them overnight and performed alkaline lysis at 95ºC during 3 min. Lysates were centrifuged during 3 minutes and 1ul supernatant was used to amplify each insert with Expand Long Template PCR system (Roche). 83% of reactions were positive and average insert size was approximately 4 Kb. Then, we purified inserts with a 96-well plate DNA purification kit (Telechem Intl.), dried samples, resuspended in Microspotting solution plus (Telechem) and finally reorganized samples into 384-well plates, using a liquid handling machine (eppendorf). Finally, arrays were spotted with MicroGrid II TAS arrayer.