Phage ØVC8 DNA was extracted as follows: a 500-μl aliquot of phage ØVC8 suspension (1 × 108/ml PFU) was treated with 100 U of DNase I (Invitrogen, Carlsbad, CA, USA), 2 μl of proteinase K (20 mg/ml) (Vivantis, Oceanside, CA, USA), and 50 μl of SDS (10 %) at 56 °C for 1 h. The purified DNA was treated with phenol-chloroform (1:1 ratio), precipitated with cold absolute ethanol, and resuspended in DNase-free water.
Proteinase k
Manufactured by Vivantis Technologies
Sourced in Malaysia
Proteinase K is a broad-spectrum serine protease used in various laboratory applications. It is effective in digesting and denaturing proteins, enabling DNA and RNA extraction and purification. Proteinase K is widely used in molecular biology, biochemistry, and genomic research.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Ultra tech hrp kit, by Beckman Coulter (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse antibody, by Agilent Technologies (1 mentions)
Lysozyme, by Vivantis Technologies (1 mentions)
Gf 1 bacterial dna extraction kit, by Vivantis Technologies (1 mentions)
Xylene, by Merck Group (1 mentions)
5 protocols using proteinase k
1
Phage ØVC8 DNA Extraction Protocol
2
Stability Assessment of Anti-MRSA Peptide
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The stability of the anti-MRSA peptide in response to temperature, pH, and proteolytic enzyme was determined by incubating the sample solutions (80 µg/mL) in the following conditions. Heat treatment was conducted at 25, 50, 75, and 100 °C for 1 h. The sensitivity to acid and basic solutions was performed at different pH of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 for 2 h at 37 °C. The pH was subsequently adjusted to 7.0 before further evaluation. Sensitivity to proteolysis was tested by incubating the purified peptides with trypsin, α-chymotrypsin, and proteinase K (1.0 mg/mL) (Vivantis Technologies Sdn. Bhd., Selangor Darul Ehsan, Malaysia) at 37 °C for 2 h. The antimicrobial activity of all treated samples was determined by agar well diffusion against S. aureus TISTR 517 and MRSA isolate 2468. The residual activity was calculated as a percentage based on the proportion of the inhibition zone of treated and untreated samples. The untreated peptides and fresh medium were used as the negative and blank controls, respectively (Baindara et al., 2016 (link)). Each experiment was conducted in triplicates, and the data were presented as mean ± SD. The result analysis was done by Student’s t-test, and the p-value of less than 0.05 accounted for statistical significance.
3
Immunohistochemical Analysis of Engineered Cartilage
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The expression of COL II in the engineered cartilage was examined by immunohistochemical staining at 12 and 24 weeks after implantation. Briefly, paraffin sections were deparaffinised followed by hydration in ethanol solutions of decreasing concentrations (100% to 70%), and pre-treated with 0.1% of Proteinase K (Vivantis Technologies, Oceanside, California) at room temperature for 30 minutes. The sections were then treated with protein blocking agent ( UltraTech HRP Kit, Beckman Coulter France, Villepinte, France) for ten minutes and incubated with goat serum to block non-specific sites. The slides were washed in PBS three times and the sections were incubated at 37°C for one hour with mouse anti-collagen type II monoclonal antibody diluted in PBS (1:200), followed by incubation with 1:100 diluted horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Dako, Carpinteria, California) for 30 minutes. Colour was developed with diaminobenzidine tetrahydrochloride (DAB).
4
Bacterial Genomic DNA Extraction Protocol
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Pure genomic DNA was obtained using the GF-1 Bacterial DNA Extraction Kit (Vivantis, Selangor, Malaysia). LAB isolates were grown in MRS or M17 broth for 24 h at 37 °C, and 3 mL of bacterial culture was centrifuged at 10,000× g for 2 min to obtain a pellet [21 (link)]. The pellet was resuspended in 80 µL of R1 Buffer and treated with 20 µL of lysozyme (50 mg/mL; Vivantis, Selangor, Malaysia) for 30 min at 37 °C. The cells were centrifuged at 10,000× g for 3 min to form the pellet, which was resuspended in 180 µL of R2 Buffer and 3 µL of Proteinase K (10 mg/mL; Vivantis, Selangor, Malaysia) and incubated in a dry bath at 65 °C for 40 min. Then, 3 µL of RNAse A (20 mg/mL; Vivantis, Selangor, Malaysia) was added and incubated at 37 °C for 10 min, and 372 µL of BG Buffer homogeneous solution was added to the sample and incubated at 65 °C for 20 min. The washing of the DNA was conducted with a clean glass filter membrane, to which absolute ethanol (200 µL) and the sample (558 µL) were transferred and centrifuged at 10,000× g for 1 min. The membrane was washed with 650 µL of Wash Buffer and centrifuged at 10,000× g 1 min two times for removal of residual ethanol. Pure DNA in the membrane was eluted in 30 µL pre-heated TE buffer for 2 min, centrifuged at 10,000× g for 2 min, and stored at −20 °C, as recommended by the manufacturer.
5
Paraffin-embedded tissue de-paraffinization
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Using a microtome, 10 cuts of 5μm of each sample were prepared. The sample cuts were collected in a 2ml RNase-free microtube for de-paraffinization. De-paraffinization was then started by adding 1ml Xylene (Merck, Germany), which continued by vortexing for 5 seconds. The samples were subsequently put in a rotator for 30 minutes and then centrifuged at 12000 rpm/15 minutes. After discarding the supernatant, 1ml of ethanol was added. Next, the samples were incubated at room temperature for 5 minutes. Then, they were centrifuged at 12000 rpm/15 minutes. Removing the supernatant, 30μl of acetone was added to each sample and left to dry. Afterwards, the samples were digested by 10μl of Proteinase K (Vivantis, Malaysia) enzyme and 200μl of enzyme buffer at 37 o C left overnight. Finally, 10μl of Proteinase K enzyme was added to the samples and incubated at 50 o C for 3 hours and 10 minutes at 72 o C.
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