Takara la taq hot start version

Semiquantitative PCR analysis was carried out as previously described using cDNA samples described above (Nomaguchi et al., 2016 (link)). Briefly, PCR products were made by using TaKaRa LA Taq Hot Start Version (Takara Bio) and a specific primer set for vpr mRNAs (Figure 1). As controls, all HIV-1 mRNA species and GAPDH mRNA were amplified using specific primer sets (Figure 1) (Nomaguchi et al., 2016 (link)) in parallel with vpr mRNAs. PCR products were separated on gels prepared by MetaPhor agarose (Lonza), and stained with ethidium bromide. Signal intensities of PCR products were measured by Amersham Imager 600 instrument (GE Healthcare).

Long‐range PCR of GD‐395 was performed on 2720 Thermal Cycler (Applied Biosystems) using the TaKaRa LA Taq^® Hot Start Version (Takara Bio, Otsu, Japan). Approximately 50 ng of high quality template DNA was added to a 15 ul standard reaction. The forward primer (TMPRSS3_In2_F) and the reverse primer (TMPRSS3_Ex12_R) were added to a final concentration of 0.67 μmol/L. Thermocycling conditions were as follows: 1 cycle of 95°C for 5 min, 30 cycles of 98°C for 20 s and 68°C for 12 min, and 1 cycle of 68°C for 7 min. Gap‐PCR was designed to detect the certain CNV in single PCR amplification for GD‐395 and his family members. One reverse primer (TMPRSS3_Ex12_R) and two forward primers (TMPRSS3_In2_R and TMPRSS3_Ex11_F) were added to a single gap‐PCR amplification. Standard protocols of Sanger sequencing were followed on the ABI 3500xL Dx Genetic Analyzer (Applied Biosystems) to confirm detected variants in cases and extended families.

A DNA fragment containing the X. tropicalis DS-3′ was amplified using TaKaRa LA Taq Hot Start Version (TaKaRa) and the primers XhoXtDS5 (5′-GGCTCGAGTAGGTGTGGCAGCACAA-3′) and XtDS3BamHI (5′-GGGGATCCGAATTTCCCTAAATTGTCTTTACAAAG-3′) in a three-step protocol (94°C for 60 seconds; 35 cycles of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and 72°C for 10 minutes). The product was digested using XhoI and BamHI and then ligated into pEGFP-C3 (Clontech). The presence of the DS-3′ was confirmed by DNA sequencing. This construct was designated pEGFP-DS. The DS-3′ fragment was inserted into the XbaI site of pCS2-Flag-TALEN-ELD/KKR (Lei et al., 2012 (link)) using the In-Fusion Advantage PCR cloning kit to obtain pTALEN-ELD/KKR-DS. The DNA-binding domains were designed to target the first exon of the tyrosinase gene (Tyr-B) (Nakajima et al., 2013 (link)). TALEN repeats were assembled as previously described (Cermak et al., 2011 (link)), with minor modifications (Nakajima et al., 2013 (link)), and were inserted into pTALEN-ELD-DS and pTALEN-KKR-DS to generate the Tyr-TALEN-DS expression constructs. The target sequences of Tyr-TALEN-ELD and -KKR were 5′-GGCCCTCAGTTTCCAT-3′ and 5′-GGCCAGTTCTCTCTAT-3′, respectively.