Longamp high fidelity taq dna polymerase

In order to generate a recombinant PP5 protein, a 1.4 kb region containing PP5 was amplified from genomic DNA of L. major FV1 using the primers 5′-ACC CTC GAG ATG GAG GAG TCC GAC CGC-3′ (XhoI), R 5′-GCC GCG GCC GCT TAA AAT AGA CCC GCG CC-3′ (NotI) and LongAmp high-fidelity Taq-DNA polymerase (New England Biolabs) following the manufacturer’s recommendation. The product was cloned into pGEM-T (Promega) to create pGEM-T-PP5. N-terminal GST-PP5 fusions (Glutathione S-Transferase) were obtained by inserting the 1.4 kb PP5 fragment from pGEM-T into the respective site of pGEX-5x. pGEX-5x-PP5 constructs were then transformed in BL-21 Escherichia coli (E. coli) cells (NEB). Cells transfected with the empty vector, pGEX-5x, were used as mock controls.

Total RNA was isolated from L. major and L. donovani WT and transgenic parasites with Trizol reagent (Life Technologies Inc., NY) using RNase-free plastic supplies. cDNA was amplified using M-MLV Reverse Transcriptase (Sigma) and oligo d(T)₁₅ (Promega) following manufacturer's recommendations. PCR was carried out with LongAmp high fidelity Taq- DNA polymerase (New England Biolabs) using LmaPA2G4 specific primers 5′- CCACGTGGACGGCTACTGCGCCG-3′ and 5′- CTTCCTTTTCGAAGAGAATAGGG-3′ and GAPDH primers 5′- CGACGACGGCAAAGCAGAAG-3; and 5′- TCAGCGCCACACCGTTGAAG-3′. All RNA samples were treated with DNA-free (Ambion, Inc., TX) to remove contaminating genomic DNA. Each RT-PCR product was analyzed by gel electrophoresis using 1% agarose gels and band intensity analyzed with ImageQuant TL software (GE Healthcare).