Ingenius gel documentation system

Following the removal of the ficoll (Sigma-Aldrich, MO) gradient fraction containing the PBMC from 5 ml of whole blood, the remaining red blood cell-containing fraction was culture in liver digested-neutralized tryptose (LDNT) medium at 27° C for up to 3 months. Cultures were periodically screened under inverted microscope for motile parasites. Cultures with detectable parasites were propagated in order to obtain and characterize T. cruzi isolates. Cultures with no visible parasites were subjected to standard PCR. Briefly, total DNA from hemocultures was extracted using a blood DNA extraction kit (Qiagen, MD) and amplified with OneTaq 2x MasterMix solution (New England Biolabs, MA) mixed with T. cruzi specific primers for satellite DNA [48 (link)]. PCR products were run on 1.5% agarose gels and imaged in an InGenius gel documentation system (SynGene, MD).

PCR amplification of lung SOD and CAT was performed using specific pair of primers (synthesized by Macrogen Co., GAsa-dong, Geumcheon-gu., Korea) for rat. The sequences of specific primers and product sizes are listed in Table 2.
4 microliters of cDNA and 1.25 μM of each primer were added to 12.5μM 2xGoTaq PCR master mix (Promega Corporation, Madison, WI, USA). The volume was completed to 25 μl with DNase free water and samples were loaded into Techne TC-3000x thermal Cycler (Bibby Scientific, England). PCR conditions were 94 C for 5 mins followed by cycles of 1 min at 95°C, 1 min at annealing temperature 60°C and 1 min at 72°C. The cycle number was adjusted so that all reactions were within the linear range of product amplification according to the reference gene (28 cycles). The final extension step was 7 min at 72°C, PCR products were separated on 1.5% agarose-A (Bio Basic, Markham, ON, Canada) gel in 1.0 X-TAE (Tris–Acetate-EDTA) buffer (Sigma–Aldrich, St. Louis, MO, USA) at 100 V for 30 min.
The gel was stained with ethidium bromide (Sigma–Aldrich), visualized and photographed under UV light using Ingenius gel documentation system (Syngene Europe, Cambridge, UK) [20 ].