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[…] ctober 16, 2012, protocol #15-047)., Leishmania major strain Friedlin V1 (MHOM/JL/80/Friedlin) was cultured in M199 medium supplemented with 10% FBS at 26°C and pH 7.4 . L. donovani strain 1S2D (MHOM/SD/62/1S-CL2D) was grown in M199 supplemented with 10% FBS and axenic amastigotes were differentiated as described previously . For some experiments L. major metacyclic promastigotes were enriched by agglutination . Briefly, cells were incubated for 30 min at RT with 50 µg/ml peanut agglutinin in M199 without serum, agglutinated parasites were removed by centrifugation and metacyclic parasites were recovered from the supernatant., L. major CAJ07101 (gi|68126048) was used as an initial query for PSI-BLAST and after four cycles results with significant E-values (<10e−6) were selected. Sequences corresponding to putative aminopeptidase proteins from the sequenced genomes of H. sapiens, L. major, L. infantum, L. braziliensis, L. donovani, T. brucei, T. vivax, T. cruzi and T. congolense were retrieved using TriTrypDB and UniProt databases; (http://tritrypdb.org/tritrypdb/) and (www.uniprot.org). Sequences were aligned with Clustal X (v 2.0). Alignments were converted to MEGA compatible files and fed into the MEGA5.2 software package. A Neighbor-Joining tree was computed with 500 bootstrap replicates., In order to generate null mutants, a 901 bp region in the 5′ untranslated region (UTR) upstream of LmaPA2G4 was amplified with the primers 5′-ACCGGTACCCAATCATGGCCCACCGAAGG- 3′ (KpnI) and 5′-CGCCCCGGG/CTCGAGTTTTTTTGGGTGGGTGGC-3′ (SmaI or XhoI). A 903 bp fragment in the 3′UTR was amplified with primers 5′-ACCCTCGAG/GGATCCACGGCCGTGGCATCCGTG-3′ (XhoI or BamHI) and 5′-CGCGGTACCCACGATGGGCAGAACGCC- 3′ (KpnI). Reactions were performed in a total volume of 50 µl containing LongAmp high fidelity Taq- DNA polymerase (New England Biolabs) following the manufacturer's recommendation. Products were cloned into p […]

Pipeline specifications

Software tools BLASTP, Clustal W, MEGA
Databases TriTrypDB