Fig. 4

PCR, cloning and sequencing. Total DNA was extracted from 100 mg paraffin-embedded cerebral tissue using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). DNA was quantified in a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), obtaining an E. histolytica 128 bp amplicon for the rRNA gene, which was cloned with the CloneJET PCR Cloning Kit (Thermo Scientific) using a pJET1.2/blunt cloning vector. Then, the ligation mixture was used for transformation of Escherichia coli DH5a calcium-competent cells. Plasmid DNA was extracted from heat-shocked cells with the Zyppy Plasmid Miniprep (Zymo Research, Irvine, CA, USA). Clones were analyzed by PCR to verify the insertion of the amplicon into the pJET1.2/blunt vector. The plasmid sequence shows forward and reverse primers (electropherograms) that correspond to the E. histolytica rRNA gene sequence. Hu = 120 bp amplicon for human β-actin; M = bp marker; Eh = 128 bp amplicon for the E. histolytica rRNA 18 s gene, NTC = no template control