Proteinase k

Genomic DNA was purified from Replic cells and chopped mouse organs incubated with proteinase K (Nakalai Tesque) overnight, and bisulfite conversion of genomic DNA was performed using an EpiTect Bisulfite Kit (Qiagen). Bisulfite-converted DNA of the Epo and Epas1 gene promoters was amplified by PCR using primers listed in Supplementary Table 1. Subsequently, TA cloning of the amplified genomic DNA fragments into the pT7Blue T-vector (Novagen) was conducted. Eight clones for each promoter were bilaterally sequenced by using primers (Supplementary Table 1) annealing to the cloning site of the pT7Blue T-vector.

To examine the effect of temperature, the crude extract (5 mg/ml) was incubated at 4, 10, 28, 37, 60, 70, 80, 90, and 100 °C for 6 h respectively. After the sample was cooled to room temperature, the residual antifungal activity of the treated sample (50 µl) was tested by agar-spot assay using A. brasiliensis NBRC9455.
To evaluate the pH stability of the purified antifungal compound, pH values of the crude extract (5.0 mg/ml) were adjusted from 2.0 to 12.0 at intervals of 2.0 by substitution of each solvent by ultrafiltration, and the adjusted crude extract kept at 4 °C for 12 h. Afterward, the pH values were readjusted to 7.0, and the residual antifungal activity of the treated sample (50 µl at the final concentration of 5.0 mg/ml-distilled water) was tested by the agar-spot assay using A. brasiliensis NBRC9455.
For evaluation of the effect of proteinase K, the crude extract (5.0 mg/ml-distilled water) was treated with 1.7 mg of proteinase K (56 unit, Nacalai Tesque, Inc., Kyoto, Japan) at 37 °C for 30 min. The residual antifungal activity of the treated sample (50 µl) was tested by an agar-spot assay using A. brasiliensis NBRC9455. As positive and negative controls, the crude extract not treated with proteinase K and proteinase K solution not containing the crude extract were used, respectively, in the agar-spot assay.

Supercoiled, relaxed, linear pBR322 plasmid DNAs as well as kDNA were purchased from Inspiralis (Norwich, UK). AMPPNP, SYBR Gold and NativeMark™ unstained protein standard were purchased from Thermo Fisher Scientific (MA, USA). Proteinase K was purchased from Nacalai Tesque (Kyoto, Japan).

PCR analysis was performed on electroporated cells, foetal F₀ clones, and F₁ and F₂ progeny. Electroporated cells were digested with proteinase K (Nakarai Tesque) solution and the mixture was used as a template: from ear tissues of foetuses and piglets, genomic DNA was prepared as a template using a GenoPlus Genomic DNA Extraction Miniprep System (Viogene, Taipei, Taiwan)^{17 (link),38 (link),56 (link)}. In both cases, PCR amplification was performed with LA Taq Hot Start Version (Takara Bio, Otsu, Japan), using the primers Neo sF: 5′-CGCCTTCTTGACGAGTTCTTCTG-3′ and Exon 22 sR: 5′-TAAGGTGCCCGTGGAATTCCCTC-3′^{17 (link)} (defined as Neo-Ex 22 PCR) (Fig. 2). Thermal cycling parameters were: 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, then 8 min at 68 °C, followed by a final step of 7 min at 68 °C. This amplification yields a mutant-allele-specific product of 6.0 kb.

Traditional DI and piggyBac TS were analyzed by the same method. Expression of EGFP fluorescence in newborn transgenic mice was visualized by UV microscopy (VB-6000; KEYENCE Inc., Osaka, Japan) (Fig. 6). PCR genotyping of offspring was performed using a crude lysate of the tail tips (Fig. S4). Genomic DNA was extracted from the mouse tail using Direct PCR Lysis Reagent (Viagen Biotech Inc., Los Angeles, CA, US) containing 0.2 mg/ml proteinase K (Nacalai Tesque Inc., Kyoto Japan). The DNA lysate was used as a template in a 20-µl PCR reaction volume using go-Taq premixture (Promega Corporation) in the presence of the following primers to amplify the EGFP transgene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. EGFP forward 5′-GCGACGTAAACGGCCACAAG-3′, EGFP reverse; 5′-TAGGTCAGGGTGGTCACGAG-3′. GAPDH forward 5′-ACCACAGTCCATGCCATCAC-3′, GAPDH reverse; 5′-TCCACCACCCTGTTGCTGTA-3′.

To investigate the factors that contribute to biofilm degradation, we performed an enzyme treatment on the formed biofilm. Enzymatic solutions with a final concentration of 5 μg/mL of proteinase K (Nacalai Tesque), α-amylase (Wako Pure Chemical Industries Co. Ltd.), or lipase (Nacalai Tesque) in Tris-HCI buffer pH 7.0 were prepared. Each enzymatic solution was then added to wells containing biofilm, as a single or combination enzyme mixture and incubated under static anaerobic conditions at 37°C for 24 h. Biofilm measurements were performed as previously described. Biofilm without this treatment was used as a control.