Nebnext high fidelity 2x pcr master mix

For overexpression of sorghum SbKOR1 gene in Arabidopsis (Col-0), the predicted 1863 bp coding sequence (CDS) of SbKOR1 gene was amplified from sorghum wild type BTx623 cDNA library using NEB Next high-fidelity 2X PCR Master Mix (New England Biolabs, Ipswich, MA, USA). The PCR product was then digested with AscI and PacI (New England Biolabs, Ipswich, MA, USA) restriction enzymes and sub-cloned into the AscI and PacI digested pMDC32 binary vector. For complementation, the predicted 3 kb of AtKOR native promoter was amplified from Arabidopsis thaliana (Col-0) using NEB Next high-fidelity 2X PCR Master Mix (New England Biolabs, USA). The PCR product was then digested with SbfI and AscI (New England Biolabs, USA) and sub-cloned into the SbfI and AscI digested pMDC32 binary vector containing SbKOR1 genes. The digested product of pMDC32 binary vector with 1863 bp CDS of SbKOR1 gene was ligated with digested KOR native promoter from Arabidopsis (Col-0) 3kb. The over-expression construct was confirmed by Sanger sequencing (GenewizTM, South Plainfield, NJ, USA). The gene fragments of the overexpression construct were PCR amplified using (Supplementary Table S1) specific primers, gel purified, and sequenced. The sequencing results were analyzed by using BLASTN.

First, the amount of genomic DNA (gDNA) needed for adequate coverage was determined by calculating the “library complexity x experimental coverage × 6.6 pg”. The PCR amplification was performed in two steps. The calculated total amount of gDNA was used in the first PCR (PCR1) as 2 µg of gDNA for each reaction which was set up in 50 µl with 25 µl of NEBNext High-Fidelity 2X PCR Master Mix, 2.5 μl of each primer (10 μm) and water. The thermal cycler parameters were set as follows: the initial denaturation at 98 °C for 5 min, 20 cycles of denaturation at 98 °C for 50 s, annealing at 68 °C for 45 s, and elongation at 72 °C for 1 min, and final extension at 72 °C for 5 min. All PCR products were then pooled and mixed in the second PCR (PCR2). 25 µl of the PCR1 product was added into the PCR2 reaction together with 50 µl of NEBNext High-Fidelity 2X PCR Master Mix, 5 μl of each primer (10 μl) which contains Illumina adaptors and index sequences, and water up to 100 µl. The PCR2 was set as follows: initial denaturation at 98 °C for 3 min, 10 cycles of denaturation at 98 °C for 50 s, annealing at 68 °C for 45 s, and elongation at 72 °C for 45 s, and final extension at 72 °C for 5 min. PCR2 product was purified from 1% agarose gel and sequenced as described for the plasmid library. Primer sequences for PCR1 and PCR2 for all library constructs are listed in Supplementary Table S4.

A 50 μl PCR reaction using NEBNext® High-Fidelity 2X PCR Master mix (New England Biolabs®) using the 5 ng-1 μg total DNA template reaction master mix protocol [25 μl of NEBNext® High-Fidelity 2X PCR Master mix (New England Biolabs®), primer pair mix 20 μl (25 μM concentration) and 23 μl of extracted DNA] was assembled. The work was performed under a UV hood in DNA free cabinet and the PCR mix was immediately transferred to a thermocycler (M J Thermocycler) preheated to 98 °C. The PCR cycling conditions were 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, annealing temperature of 60 °C for V1-V3 primer pair or 56 °C for V3-V4 primer pair for 30 s and 72 °C for 30 s, and 72 °C for 10 mins. These thermocycling conditions were based on the optimization work on the PCR.