Orf-I sequencing was performed as described previously36 (link) with slight modifications.
Approximately 1 × 106 cells were collected and DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen), according to manufacturer’s instructions. DNA fragment covering orf-I was amplified by PCR using Platinum PCR SuperMix (Invitrogen) with primers (p12-F: 5′-CACCTCGCCTTCCAACTG-3′, p30-R: 5′-GGAGTATTTGCGCATGGCC-3′) and 300 ng of genomic DNA. The PCR reaction was carried out using an Eppendorf Master Cycler gradient PCR machine, for 35 cycles of; 94°C for 30 sec, 55°C for 30 sec, and 68°C for 50 sec. The PCR product was purified using Qiaquick PCR purification kit (Qiagen) and 50 ng DNA together with 0.64 picomol of primer (5′-CTGGACAGGTGGCCAGTA-3′) was used for sequencing. The Sanger sequencing was carried out at the CCR genomics core at NCI, NIH.
Approximately 1 × 106 cells were collected and DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen), according to manufacturer’s instructions. DNA fragment covering orf-I was amplified by PCR using Platinum PCR SuperMix (Invitrogen) with primers (p12-F: 5′-CACCTCGCCTTCCAACTG-3′, p30-R: 5′-GGAGTATTTGCGCATGGCC-3′) and 300 ng of genomic DNA. The PCR reaction was carried out using an Eppendorf Master Cycler gradient PCR machine, for 35 cycles of; 94°C for 30 sec, 55°C for 30 sec, and 68°C for 50 sec. The PCR product was purified using Qiaquick PCR purification kit (Qiagen) and 50 ng DNA together with 0.64 picomol of primer (5′-CTGGACAGGTGGCCAGTA-3′) was used for sequencing. The Sanger sequencing was carried out at the CCR genomics core at NCI, NIH.
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