HEK293T cells (obtained from ATCC and preserved by our laboratory) were maintained in Dulbecco’s Modified Eagle Medium high glucose (HyClone) supplemented with 10% fetal bovine serum (Gibco). Cells were seeded in a 24-well plate at a density of approximately 1.6 × 105 cells per well to reach 70–90% confluence and transfected with 50 µL serum-free Opti-MEM (Gibco), which contained 3 µg polyethylenimine (PEI) (Sigma-Aldrich), 0.3 µg sgRNA expression vector and 0.7 µg base editor expression vector. Cells transfected with 50 µL serum-free Opti-MEM, which only contained 3 µg PEI (without vector), were served as untreated control. About 60 h after transfection, EGFP+ cells were sorted by FACS AriaIIU (Becton Dickinson), given that all base editor expression vectors contain EGFP fluorescence labels, and subjected to lysis in 10 µL lysis buffer with 1% NP40 (MP Biomedicals) and 50 µg mL−1 proteinase K (Tiangen).
Proteinase k
Manufactured by Tiangen Biotech
Sourced in China
Proteinase K is a serine protease enzyme that is commonly used in molecular biology and biochemistry laboratories. Its core function is to degrade and break down proteins by cleaving peptide bonds. Proteinase K is widely used for various applications, including DNA and RNA extraction, protein purification, and sample preparation for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rnase a, by Tiangen Biotech (5 mentions)
Dnase 1, by New England Biolabs (3 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (3 mentions)
Histiocyte lysis buffer, by Tiangen Biotech (2 mentions)
T7 sp6 rna transcription kit, by Roche (2 mentions)
Dimethyl sulfoxide (dmso), by Yuanye Bio-Technology (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facsaria iiu, by BD (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Ddh2o, by Tiangen Biotech (1 mentions)
Antifade mounting medium, by Boster Bio (1 mentions)
Paraformaldehyde fix solution, by Wuhan Servicebio Technology (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Phosphatase inhibitor, by Selleck Chemicals (1 mentions)
Mito tracker red cmxros, by Beyotime (1 mentions)
Dnase 1, by Beyotime (1 mentions)
Cell mitochondrial stress test kit, by Agilent Technologies (1 mentions)
24 protocols using proteinase k
1
CRISPR Base Editor Transfection in HEK293T
2
Genomic DNA Extraction from Plant Tissue
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For all samples, genomic DNA was extracted from 100 mg powder using the phenol/chloroform method [19 ]. Briefly speaking, 800 μL histiocyte lysis buffer (Tiangen, China) with 100 μg proteinase K (Tiangen, China) was added to each sample, vortexed, and incubated at 65°C for 60 min with occasional vigorous shaking; an equal volume of phenol/chloroform was added, mixed, and centrifuged at 12000 rpm for 10 min. The aqueous (upper) layer was transferred to a clean tube; an equal volume of chloroform was added, mixed, and then centrifuged for 5 min at 12000 rpm. The aqueous (upper) layer was transferred to a clean tube; a one-tenth volume of 3 M Na acetate (pH 5.2) and two volumes of ice-cold EtOH (100%) were added, mixed, and incubated at −20°C for 30 min and then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was removed and the DNA pellet was washed twice with 75% EtOH and centrifuged at 12000 rpm for 2 min at 4°C; the supernatant was removed and the pellet was air-dried for 30 min at room temperature, resuspended in 100 μL ddH2O, and stored at −20°C.
3
Candida auris Cell Treatment for Analysis
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The nonaggregative/yeast-form or aggregative-form cells of C. auris were incubated at 30°C for 24 h. Cells were harvested and washed 3 times with 1 x PBS. Fungal cells were resuspended in 1 mL 1 x PBS and then treated with ddH2O, proteinase K (at a working concentration of 50 μg/mL) (TianGen Biotech, Beijing, China), or trypsin (at a working concentration of 0.25% (Gibico, Inc., Beijing) at 37°C for 1 h.
4
Measuring Bacterial Aggregate Sizes
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Overnight cultures of the pprB overexpression (in pJN105) or wild-type strains were diluted 100× in FABSgen medium supplemented with 0.02% (wt/vol) l -arabinose and grown to the exponential phase (OD600 of ∼0.6) at 37°C. To measure the size of bacterial aggregates, 200 μl of each bacterial suspension was transferred to a 4-channel dish (D35C4-20-1-N; Cellvis) and left to stand for 10 min at room temperature. The bacterial aggregates were monitored under a bright-field microscope equipped with a 60× oil lens objective. One hundred images containing at least 200 bacterial aggregates were obtained every time. Three parallel experiments were conducted for each sample. The sizes of aggregates were recorded using ImageJ software. For proteinase treatment, 20 μl proteinase K (R7012; Tiangen) was added to 1 ml bacterial culture at exponential phase and incubated for 30 min at 37°C.
5
Preparation and Storage of TOMA Solution
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Dimethyl sulfoxide (DMSO) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Proteinase K was bought from Tiangen Biotech Co., Ltd. (Beijing, China). Antifade Mounting Medium was bought from Boster Biological Technology Co., Ltd. Paraformaldehyde fix solution at a concentration of 4% was obtained from Servicebio Technology Co., Ltd. (Wuhan, China). An RAA fluorescence kit was obtained from Jiangsu Qitian Gene Biotechnology Co., Ltd. (Wuxi, China). A Wizard® Magnetic DNA Purification System for Food was purchased from Promega Biotech Co., Ltd. (Madison, WI, USA). All primers and probes were synthesized by Sangon Biotech (Shanghai, China). Skim milk was purchased at the local supermarket (Rainbow, China). TOMA was provided by Ningbo International Travel Healthcare Center (Ningbo Customs Port Outpatient Department). The final concentration of TOMA dissolved in 20% DMSO was 1 mg/mL. It was stored at −20 °C in the dark for further use.
6
MERS-CoV nsp1 Antibody Generation and Cellular Assessment
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By cooperating with KWINBON (Beijing, China), mouse monoclonal anti-MERS-CoV nsp1 antibody was prepared in our laboratory; Rabbit monoclonal anti-LAMP1 antibody (6502, CST); Rabbit polyclonal anti-RPS18 antibody (A11687, Abclonal); Rabbit polyclonal anti-LSM14A antibody (A16682, Abclonal); Rabbit polyclonal anti-G3BP1 antibody (A14836, Abclonal); Rabbit polyclonal anti-COX4I1 antibody (A6564, Abclonal); TRIzol™ Reagent (15,596–026, Invitrogen); RNase inhibitor (R0102, Beyotime); Protease inhibitor (B14001, bimake); Phosphatase inhibitor (B15001, bimake); DNase I (D7073, Beyotime); Proteinase K (TIANGEN, RT403); poly(I:C) (APExBIO, B5551); D-galactosamine (Beyotime, ST1213); Tetracyclin (T8180, Solarbio); Mito-Tracker Red CMXRos (Beyotime, C1071S); Cell Counting Kit-8 (CCK-8) (K1018, APExBIO); Cell Cycle Analysis Kit (CY2001, Tianjin Sungene Biotech Co, Ltd); Cell Mitochondrial Stress Test Kit (103,015–100, Agilent Seahorse); Mitochondrial membrane potential assay kit with JC-1 (C2006, Beyotime).
7
PAXYB1 Phage Genome Sequencing
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The purified phage sample was treated with DNase I (New England Biolabs) and RNaseA (Tiangen Biotech) for 2 h at 37 °C to digest the exogenous DNA and RNA. The preparation was then treated with proteinase K (Tiangen Biotech) for 15 min at 55 °C. The phage genome DNA was further prepared with a TIANamp Bacteria DNA Kit (Tiangen Biotech). Restriction enzyme digestions of the genome DNA were performed according to the manufacturer’s instructions (Fermentas). DNA fragments were purified from agarose gels using a Gel Extraction Kit (Omega). The PAXYB1 genomic DNA was sequenced using an Illumina HiSeq. 2500 sequencer, and reads were assembled into a whole genome using SOAPdenovov2.04 software and GapCloserv1.12.
8
Chromatin Pulldown of Biotinylated saRNAs
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Mutated or wild-type HEK293A cells were cultured in 15-cm dishes and saRNAs with biotin labelled on the 3′-end of the sense or antisense strand (Ribo) were transfected at 20 nM. Streptavidin beads (Dynabeads® MyOne™ Streptavidin C1, Invitrogen) were washed and re-suspended as suggested by the manufacturer. Cells were harvested 48 h after transfection. The chromatin pull-down assay was modified from a reported protocol (35 ). The cell lysate was sonicated and centrifuged; the supernatant was retained and the DNA input was removed. Fifty microlitres of beads were added to each sample and incubated at 37°C for 1 h with shaking. Samples were treated with RNase A (Tiangen) and proteinase K (Tiangen) to remove contaminating RNA and protein. The DNA was extracted and amplified by qPCR using two different pairs of primers.
9
Dual FISH detection of miRNA and viral RNA
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Double fluorescence in situ hybridization (dFISH) was performed in the gut of viruliferous planthopper. Prehybridization solution contained biotin-labeled miR-263a probe (1 μmol mL-1; RiboBio) and digoxigenin (DIG)-labeled RNA1 3’-EUTR probe (50 ng μL-1) for hybridization. Approximately 200 bp of the RNA probe for 3’-EUTR of RNA1 was synthesized by a T7/SP6 RNA transcription kit (Roche). The gut was dissected from viruliferous fourth-instar nymphs and fixed in 4% (wt vol-1) paraformaldehyde at 4°C overnight. After washing with PBST, the gut was digested with proteinase K (20 μg mL-1; Tiangen) at 37°C for 15 min and hybridized with the miRNA probe and viral RNA probe at 37°C overnight. The midgut was washed with 0.2× SSC at 37°C to remove nonspecific binding. An anti-DIG alkaline phosphatase-conjugated antibody (1:500) and an anti-biotin antibody (1:100) were used for signal detection. Probes for cel-miR-67-3p and rice PsbP gene (KF460579) were used in the control group. The fluorescent signal of DIG or biotin was generated by HNPP/Fast Red or fluorescein-tyramide (Perkin-Elmer, Waltham, MA, USA) and recorded using a Leica TCS SP5 confocal microscope (Leica Microsystems). Primers used for probe synthesis are listed in S1 Table .
10
ChIP Assay for Protein-DNA Interactions
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The ChIP assay was performed as described previously (28 (link), 29 (link)). Briefly, 1 × 107/mL cells were fixed in 1% formaldehyde (Biosharp, Hefei, China) for 20 min at room temperature, and 125 mM glycine (BioFroxx, China) was added to quench formaldehyde. After a 30-min incubation in ChIP lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor) on ice, the lysates were sheared by sonication using a Bioruptor Plus (Diagenode, Liège, Belgium). Cross-linked chromatin samples were incubated with the indicated antibodies or normal rabbit IgG in a rotator at 4°C overnight. Subsequently, protein A/G-conjugated agarose beads (Smart-Lifesciences, Changzhou, China) were added, and the samples were incubated overnight at 4°C in a rotator, collected, and washed three times. To elute DNA fragments, immunocomplexes were incubated with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1.0% SDS) for 2 h at 65°C. One half of the eluted immunocomplexes was saved as the immunoprecipitation sample, while the other half was treated with proteinase K (Tiangen Biotech) overnight at 55°C. Finally, the DNA was purified with a TIANamp Genomic DNA kit (Tiangen Biotech). The purified DNA was detected by qPCR. The antibodies used for ChIP and the primers used for qPCR are listed in Tables 3 and 4 .
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