proteinase k  (Qiagen)


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    QIAGEN Proteinase K
    Description:
    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
    Catalog Number:
    19131
    Price:
    96.3
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    QIAGEN Proteinase K
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    Structured Review

    Qiagen proteinase k
    QIAGEN Proteinase K
    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
    https://www.bioz.com/result/proteinase k/product/Qiagen
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis"

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33541-5

    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Figure Legend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Techniques Used: Incubation

    2) Product Images from "The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ"

    Article Title: The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00929

    Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p
    Figure Legend Snippet: Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Techniques Used: Polymerase Chain Reaction, Incubation, Isolation

    3) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    4) Product Images from "Plasmid-normalized quantification of relative mitochondrial DNA copy number"

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33684-5

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Figure Legend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Techniques Used: Incubation, Lysis

    5) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    6) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Comparison of DNA extraction protocols in spiked sputum samples. The  M. tuberculosis mc 2 7000  stock suspension was diluted and used to spike negative sputum samples. Box plots with C T  median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ®  method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    Related Articles

    Centrifugation:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: The supernatants were discarded, and pellets were processed for each extraction methods as follow: (i) Chelex® method: incubation with 200 μl of 20% Chelex® 100 resin (Bio-Rad, Richmond, CA, United States) prepared in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] (Sigma-Aldrich, Germany). .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Real-time Polymerase Chain Reaction:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: The supernatants were discarded, and pellets were processed for each extraction methods as follow: (i) Chelex® method: incubation with 200 μl of 20% Chelex® 100 resin (Bio-Rad, Richmond, CA, United States) prepared in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] (Sigma-Aldrich, Germany). .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Incubation:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: The supernatants were discarded, and pellets were processed for each extraction methods as follow: (i) Chelex® method: incubation with 200 μl of 20% Chelex® 100 resin (Bio-Rad, Richmond, CA, United States) prepared in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] (Sigma-Aldrich, Germany). .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Article Title: Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution
    Article Snippet: Briefly, mice received an intramuscular injection of the DNA complexes using pCMV-luc. .. Thirty minutes after injection, tissues were harvested and homogenized in DNAzol reagent (Thermo Fisher Scientific, Carlsbad, CA) using a handheld TissueTearor homogenizer (Biospec Products, Bartlesville, OK) or with a Pellet Pestles motor grinder (Sigma-Aldrich, St. Louis, MO) and incubated overnight with proteinase K (QIAGEN, Venlo, Netherlands) (100 μg/mL) at room temperature. .. Tissue extracts were then centrifuged for 10 min at 10,000 × g at 4°C, and total DNA was extracted from the supernatant using a Wizard DNA Prep Spin column (Promega, Fitchburg, WI) following the manufacturer’s instructions.

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of static biofilms, proteinase K (Qiagen) was added to the wells at a final concentration of 5 mg/ml after 19 h of growth, and then the reaction mixtures were statically incubated for 1 h at 37°C before the nonadherent biomass was removed and the crystal violet assay performed. ..

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. .. Static biofilm formation was assessed using the crystal violet assay as previously described ( ).

    Lysis:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: The supernatants were discarded, and pellets were processed for each extraction methods as follow: (i) Chelex® method: incubation with 200 μl of 20% Chelex® 100 resin (Bio-Rad, Richmond, CA, United States) prepared in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] (Sigma-Aldrich, Germany). .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Polymerase Chain Reaction:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: The supernatants were discarded, and pellets were processed for each extraction methods as follow: (i) Chelex® method: incubation with 200 μl of 20% Chelex® 100 resin (Bio-Rad, Richmond, CA, United States) prepared in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] (Sigma-Aldrich, Germany). .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Injection:

    Article Title: Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution
    Article Snippet: Briefly, mice received an intramuscular injection of the DNA complexes using pCMV-luc. .. Thirty minutes after injection, tissues were harvested and homogenized in DNAzol reagent (Thermo Fisher Scientific, Carlsbad, CA) using a handheld TissueTearor homogenizer (Biospec Products, Bartlesville, OK) or with a Pellet Pestles motor grinder (Sigma-Aldrich, St. Louis, MO) and incubated overnight with proteinase K (QIAGEN, Venlo, Netherlands) (100 μg/mL) at room temperature. .. Tissue extracts were then centrifuged for 10 min at 10,000 × g at 4°C, and total DNA was extracted from the supernatant using a Wizard DNA Prep Spin column (Promega, Fitchburg, WI) following the manufacturer’s instructions.

    Concentration Assay:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of static biofilms, proteinase K (Qiagen) was added to the wells at a final concentration of 5 mg/ml after 19 h of growth, and then the reaction mixtures were statically incubated for 1 h at 37°C before the nonadherent biomass was removed and the crystal violet assay performed. ..

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. .. Static biofilm formation was assessed using the crystal violet assay as previously described ( ).

    Crystal Violet Assay:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of static biofilms, proteinase K (Qiagen) was added to the wells at a final concentration of 5 mg/ml after 19 h of growth, and then the reaction mixtures were statically incubated for 1 h at 37°C before the nonadherent biomass was removed and the crystal violet assay performed. ..

    Imaging:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. .. Static biofilm formation was assessed using the crystal violet assay as previously described ( ).

    Confocal Laser Scanning Microscopy:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. .. Static biofilm formation was assessed using the crystal violet assay as previously described ( ).

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    Qiagen proteinase k
    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with <t>proteinase</t> K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/Qiagen
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    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Journal: Scientific Reports

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    doi: 10.1038/s41598-018-33541-5

    Figure Lengend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Article Snippet: The medium containing the filtrate was either untreated (OS7 filtrate), or treated either with 100 μl of proteinase K (Qiagen; > 600 mAU/ml; one mAU releases 1 µmol tyrosine per min from hemoglobin at 37 °C, pH7.5) at 50 °C for 20 min followed by the heat treatment at 100 °C for 5 min (OS7 filtrate + K), or without Qiagen proteinase K at 50 °C for 20 min (OS7 filtrate +50 °C).

    Techniques: Incubation

    Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Journal: Frontiers in Microbiology

    Article Title: The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ

    doi: 10.3389/fmicb.2018.00929

    Figure Lengend Snippet: Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Article Snippet: For a more precise active component determination, heat killed bacteria were digested with 1 mg/ml Proteinase K (Qiagen, Hilden, Germany) for 1 h at 37°C.

    Techniques: Polymerase Chain Reaction, Incubation, Isolation

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Journal: mBio

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    doi: 10.1128/mBio.01376-18

    Figure Lengend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Article Snippet: For proteinase K treatment of static biofilms, proteinase K (Qiagen) was added to the wells at a final concentration of 5 mg/ml after 19 h of growth, and then the reaction mixtures were statically incubated for 1 h at 37°C before the nonadherent biomass was removed and the crystal violet assay performed.

    Techniques: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Journal: Scientific Reports

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    doi: 10.1038/s41598-018-33684-5

    Figure Lengend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Article Snippet: Both buffers were used pure and supplemented with Proteinase K (at 1 mg/ml, Qiagen, Hilden, Germany).

    Techniques: Incubation, Lysis