Bigdye terminator v3.1 reaction mix

Mutation validation was performed with bidirectional sequencing of the selected sample sites. PCR reactions were prepared using 5 ng of genomic DNA, 0.4 µM oligonucleotide primers, and 0.7X Qiagen Multiplex Master Mix (Cat. no. 206145) containing HotStar Taq, buffer, and polymerase. Reactions were performed with and without QSOL PCR additive to enhance PCR and final sequence performance. Touchdown PCR was performed with the following parameters: 98°C for 5 min. and 10 cycles of 98°C for 30 sec., 72°C for 30 sec. (decreasing by 1°C per cycle), and 72°C for 1 min. The reaction then continued with 30 cycles of 98°C for 30 sec., 63°C for 30 sec., and 72°C for 1 min, followed by a final extension at 72°C for 5 min. The PCR products were purified with a 1:15 dilution of Exo-SAP, diluted by 0.6X, and then cycle-sequenced for 25 cycles using a 1/64^th dilution of BigDye Terminator v3.1 reaction mix (Applied Biosystems, Cat. No. 4337456). Finally, reactions were precipitated with ethanol, resuspended in 0.1 mM EDTA, and analyzed on AB 3730xl sequencing instruments using the Rapid36 run module and 3xx base-caller. SNPs were identified using SNP Detector software and then visually validated with Consed.

Mutation validation was performed with bidirectional sequencing of the selected sample sites. PCR reactions were prepared using 5 ng of genomic DNA, 0.4 µM oligonucleotide primers, and 0.7× Qiagen Multiplex Master Mix (Cat. no. 206145) containing HotStar Taq, buffer, and polymerase. Two reactions were completed for each sample site, one with and one without, using the QSOL PCR additive provided in the kit. Touchdown PCR was performed with the following parameters: 98 °C for 5 min and 10 cycles of 98 °C for 30 s, 72 °C for 30 s (decreasing by 1 °C per cycle), and 72 °C for 1 min. The reaction then continued with 30 cycles of 98 °C for 30 s, 63 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The PCR products were purified with a 1:15 dilution of Exo-SAP, diluted by 0.6×, and then cycle-sequenced for 25 cycles using a 1/32nd dilution of a BigDye Terminator v3.1 reaction mix (Applied Biosystems, Cat. No. 4337456). Finally, the reactions were precipitated with ethanol, resuspended in 0.1 mM EDTA, and analyzed on AB 3730xl sequencing instruments using the Rapid36 run module and 3xx base-caller. SNPs were identified and visually validated with a Mutation Surveyor.