Dnase rnase free pcr grade water

Host DNA was screened for evidence of Rickettsia using PCR assay to amplify the intergenic spacer rpmE-tRNA^fMet and the citrate synthase-encoding gene gltA using previously described primer sets shown in Table 1. The amplifications were carried out in a total volume of 25 µL reaction mix containing 1 µL deoxyribonucleotide triphosphates solution (dNTPs), 2.5 µL standard Taq buffer (Biolabs^®, New England, UK), 1 µL each of reverse and forward primers, 17.25 µL of DNase/RNase-Free^® PCR grade water (QIAGEN, Hilden, Germany), 0.25 µL of Taq DNA polymerase (Biolabs^®, New England, UK) and 2 µL of template DNA. The amplifications were performed in Applied Biosystems Veriti^® 96-well thermocycler (Applied Biosystems, California, USA). The PCR conditions were as follows: 3 min initial denaturation at 95 °C, 35 cycles of 30 s denaturation at 95 °C, 30 s primer annealing at temperatures specific for each of the primers (Table 1), one minute extension at 72 °C and a final 10 minute extension at 72 °C. The mixture was then maintained at 4 °C. A negative control using DNase/RNase-Free^® PCR grade water (QIAGEN, Hilden, Germany) was included for quality control. The positive control used was DNA extracted from Rickettsia africae isolated from a tick in Kenya. The PCR products were visualised using agarose gel electrophoresis to check for the presence of band and size of amplicon.