Bigdye v3.1 sequencing buffer

Amplicons were purified for sequencing by adding 2μl FastAP (1U/ μl) and 0.5μl (20U/ μl) ExoI enzymes (Thermo, Vilnius, Lithuania) to 19μl of the PCR product and incubated at 37°C for 15 min. This was followed by an 85°C for 15 min incubation.
Sanger sequencing reactions on 2μl of the purified PCR was by adding 1µl BigDye ® Terminator mix v3.1 (Applied Biosystems, Foster City, CA, USA), 2.25µl 5x BigDye ® v3.1 sequencing buffer, 0.75µl Univ-p33-F primer (2µM) and molecular grade water to a total volume of 10µl and using 1 cycle of 94°C for 1 min, 30 cycles of 94°C for 10
RNA isolation, reverse transcription and PCR amplification seconds, 50°C for 5 seconds and 60°C for 4 minutes. Sequencing products were purified using ethanol precipitation, according to Sambrook (2001) . The purified sequencing products were submitted to the African Centre for Gene Technologies (ACGT), Automated Sequencing Facility, Department of Genetics, University of Pretoria, South Africa and sequenced using an ABI Prism® 3500xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequences not conforming to a quality criterion of a minimum PHRED score of 30 were discarded from further analysis.

To remove single stranded DNA from PCR products, 0.5μl of 10 U exonuclease (Thermo, Vilnius, Lithuania) and 2μl of 2U FastAP® (Thermo, Vilnius, Lithuania) was added to the amplification products and reaction was carried out as per manufacturer's instructions. The amplicons were subjected to direct Sanger sequencing by adding 1µl BigDye ® Terminator mix v3.1 (Applied Biosystems, Foster City, CA, USA), 2.25µl 5x BigDye ® v3.1 sequencing buffer, 0.75µl 2µM Univ-p33-F primer and molecular grade water to a total volume of 10µl, to 2μl of the purified PCR products. A single cycle of 94°C for 1 minute, 30 cycles of 94°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes was utilised for the sequencing reaction. Sequencing products were purified using ethanol precipitation, according to Sambrook (2001) . The purified sequencing products were submitted to the African Centre for Gene Technologies (ACGT), Automated Sequencing Facility, Department of Genetics, University of Pretoria, South Africa and sequenced using an ABI Prism® 3500xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequences not conforming to a quality criterion of a minimum phred score of 30 were omitted from further analysis.

To remove single stranded DNA from PCR products, 0.5μl of 10 U exonuclease I (Thermo, Vilnius, Lithuania) and 2μl of 2U FastAP® (Thermo, Vilnius, Lithuania) was added to 19μl amplification products and reaction was carried out as per manufacturer's instructions. Sanger sequencing reactions were carried out by adding 1µl BigDye ® Terminator mix v3.1 (Applied Biosystems, Foster City, CA, USA), 2.25µl 5x BigDye ® v3.1 sequencing buffer, 0.75µl 2µM Univ-p33-F primer and molecular grade water to a total volume of 10µl, to 2μl of the purified PCR products and using 1 cycle of 94°C for 1 min, 30 cycles of 94°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes. Sequencing products were purified using ethanol precipitation, according to Sambrook (2001) . The purified sequencing products were submitted to the African Centre for Gene Technologies (ACGT), Automated Sequencing Facility, Department of Genetics, University of Pretoria, South Africa and sequenced using an ABI Prism® 3500xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequences not conforming to a quality criterion of a minimum PHRED score of 30 were discarded from further analysis or sequencing re-done.