Onetaq quick load master mix

DNA was extracted from tissue samples using the ZR Genomic DNA Tissue MiniPrep Kit according to the standard manufacturer’s protocol. The universal primer pair LCO1490 and HCO2198 (Folmer et al., 1994 (link)) was used to amplify a fragment of the mitochondrial gene cytochrome oxidase I (COI). PCR amplifications were carried out using 12.5 µl of OneTaq Quick-Load Master Mix (New England BioLabs), 9.5 µl of molecular biology grade water, 0.50 µl of forward and reverse primer (10 µM), 1 µl of 1% bovine serum albumin (BSA) and 1 µl of template DNA to make up a total reaction volume of 25 µl. Thermal cycling conditions were as follows for M. elityeni and M. sherlockaen. sp.: initial denaturation at 95 °C for 3 minutes, followed by 35 cycles of 94 °C for 20 seconds, 45 °C for 30 seconds and 72 °C for 1 minute, followed by a final extension time at 72 °C for 5 minutes. Amplicons were Sanger sequenced at the Central Analytical Facility at Stellenbosch University using just the forward primer (LCO1490). Quality control was performed on sequences to check for any sequencing errors using BioEdit (v7.2.6) (Hall, 1999 ).

ue57 fish were genotyped as follows. A 205 bp PCR product including the ue57 mutation, was amplified using the following primers: Fwd 5’-CCACTAATAAAGACTATGTTTTCTCCTTTTTGCTC and Rev 5’-CTTCAGTTTCCGACACAGTATTG. Note the bolded G in the fwd primer represents of T > G mismatch introduced to generate an MwoI restriction enzyme cut site in the wild-type DNA sequence. 10 µM of each primer was combined with OneTaq QuickLoad Master Mix (NEB #M0486) and run in a thermocycler as follows: 94^oC for 2 min; 36 cycles of 94 °C for 30 s, 51 °C for 20 s, 68 °C for 10 s; and 68 °C for 5 min. The PCR product was digested with 1 unit of MwoI (NEB #R0573S) for 2 h at 60 °C, then run on a 3% agarose gel at 150 V for 45 min. The ue57 mutation disrupts the MwoI cut site resulting in a single DNA band of ~205 bp, while wild type fish have two DNA bands of 174 bp and 31 bp.

Blood samples were collected from 72 patients taking SCB-AEDs. Genomic DNA was extracted by the standard salting-out extraction method [23] (link). Polymerase Chain Reaction (PCR) followed by sequencing for six SCN1A SNPs: rs2298771, rs3812718, rs3812719, rs2217199, rs2195144, and rs1972445 was done. Appropriate forward and reverse primer sets were prepared (Table 1). PCR conditions were: 2 μL (50–100 ng) of DNA was amplified by adding 10 μL of OneTaq® Quick-Load® master mix (NEB, UK), the reverse, and forward primers, 1.5 μL of each (10 μmol/L), and 3.5 μL of water for injection. The thermal cycler program was as follows: an initial denaturation step at 94 °C for 3 min followed by 39 cycles of denaturation at 94 °C for 30 seconds, annealing for 15 seconds at 62 °C, an extension step at 68 °C for 60 seconds, and a final extension step at 68 °C for 5 min. PCR products were visualized on agarose gel (2%) stained using RedSafe®. Sharp PCR products were good candidates for sequencing. The Sanger sequencing technique was used in our study by GENEWIZ Technical Support Group, USA (http://www.genewiz.com).

PCR was performed in a Bio-Rad T100™ Thermal cycler (Bio-Rad, Hercules, CA, USA) according to the guidelines described by Iweriebor et al. [9 (link)] for amplification of the genus-specific disulfide bond formation protein (dsbA) gene using the following universal primers: EHL dsb F 5′-GAT GAT GTC TGA AGA TAT GAA ACA AAT-3′ and EHL dsb R 5′-CTG CTC GTC TAT TTT ACT TCT TAA AGT-3′ to generate 409 bp fragments. PCR was performed in a 25 μL reaction mixture containing 12.5 μL of One Taq^® quick-load^® master mix (New England BioLabs, Ipswich, MA, USA), 1 μL of 10 pMol for each of the forward and reverse primers, 8.5 μL of nuclease-free water, and 2 μL of DNA template. The cycling conditions were as follows: an initial heating block at 94 °C for 3 min, followed by 35 cycles of denaturation at 93 °C for 30 s, then annealing at 47 °C for 30 s, with an elongation at 72 °C for 1 min and a final elongation at 72 °C for 5 min. Nuclease-free water was used as a non-DNA negative control and positive control was E. ruminantium DNA obtained from a collaborator at the Center for Zoonosis Control, Hokkaido University, Japan. PCR products were visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide at 100 volts for 45 min. Positive PCR products were sent to Inqaba Biotech (Pty), Ltd., Pretoria (South Africa) for purification and sequencing.

After reverse transcription, cDNA samples and the NRT control sample were diluted by adding 150 µL of nuclease-free water. Endpoint PCR to test for the presence of transcripts resulting from possible read-through across Rho-dependent terminators were done with a OneTaq Quick-Load Mastermix (New England Biolabs), using 1 µL of diluted cDNA or NRT control as template in a 50 µL reaction. To detect rare transcripts, we used 45 amplification cycles. As a positive control template in PCR reactions, we used 1 µL of a colony of strain MG1655 ∆tolC resuspended in 25 µL water and heated to 95°C for 4’. For agarose gel visualization, we loaded 15 µL of cDNA and NRT control PCR reactions and 2 µL of the positive control PCR reactions.