The bacterial genomic DNA was extracted with the help of HiPurA™ kit (Himedia, India). The bacterial 16S rRNA genes were amplified by PCR using two universal primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), and 1492R (5′-CGGTTAC CTTGTTACGACTT-3′) in 50µL PCR tube using 4µL each dNTP, 2µL MgCl2, 2µL template DNA, 1µL each primer (forward and reverse), 1µL Taq DNA polymerase, and 33µL nuclease-free water (Himedia, India). Reactions were performed in the MasterCycler gradient (Eppendorf, India) with the following reaction conditions; 94 °C (5 min) for early denaturation steps followed by 30 cycles of 94 °C (30 s), 55 °C (1 min), 72 °C (1 min), and extension at 72 °C (10 min). The purification of the PCR products were done by using HiPurA™ PCR clean up system kit (Himedia, India) and the sequencing was done through Applied Biosystems ABI (3500 Genetic Analyzer, Japan) using each universal primer (27F and 1492R) (Sherpa et al., 2018 (link)). The sequences were assembled and aligned with the aid of the Codon-Code Aligner software. The sequences were identified using NCBI BLASTn and the phylogenetic tree was created by using the neighbor-joining method using MEGA v.10 (Erickson, 2010 (link); Saitou and Nei, 1987 (link)). Gene bank accession was obtained post Bankit submission.
Nuclease free water
Manufactured by HiMedia
Sourced in India
Nuclease-free water is a highly purified form of water, specifically designed for use in sensitive molecular biology applications where the presence of nucleases could compromise the integrity of nucleic acids. Its core function is to provide a contaminant-free aqueous environment for various experiments and protocols involving RNA, DNA, or other nucleic acid-based procedures.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by HiMedia (2 mentions)
Ammonium persulfate, by HiMedia (2 mentions)
Loading dye, by Merck Group (2 mentions)
50 bp dna ladder, by Merck Group (2 mentions)
Transferrin a488, by Merck Group (2 mentions)
Hoechst, by Merck Group (2 mentions)
Temed, by HiMedia (2 mentions)
Mowiol, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Ethidium bromide, by HiMedia (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Taq dna polymerase, by HiMedia (1 mentions)
Hipura kit, by HiMedia (1 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Pcr readymix, by Merck Group (1 mentions)
Crystal violet, by HiMedia (1 mentions)
Sodium hypochlorite, by Merck Group (1 mentions)
Streptomycin sulfate, by Merck Group (1 mentions)
Potato dextrose agar pda, by Merck Group (1 mentions)
10 protocols using nuclease free water
1
Bacterial 16S rRNA Gene Sequencing Protocol
2
DNA Extraction from Klebsiella pneumoniae
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For DNA extraction, 62 of the randomly selected isolates exhibiting ESBL production in the initial screening test were made use of as per the procedure given by Bora et al.[15 (link)] Procedure was slightly modified to get the largest amount of DNA.
Five to six colonies of K. pneumoniae suspended in 200 μl double-distilled water and heated at 95°C for 10 min and added with equal proportion of 90% ethanol to DNA to get precipitated. The precipitated top solution was again added with equal proportion of 90% ethanol and centrifuged at 6440 × g for 5 min. Discarded the supernatant and to the deposit again added equal proportion of 90% ethanol and repeated the procedure for two more times. At the end of the procedure, the deposit was left to dry and added double distilled water to remove ethanol and again centrifuged at 6440 × g for 5 min. The deposit with few drops of nuclease-free water was directly used as template DNA for PCR study. The primers used and PCR cycling conditions were described in [Tables1 and 2 ].
The total concentration of PCR reaction to do single test was 25 μl [PCR ready mix-12.5 μl (Sigma Aldrich, Merck's Life Science), Forward primer-1 μl, Reverse primer-1 μl (Bioserve Hyderabad, India), Template DNA-4 μl and Nuclease free water-6.5 μl (Hi Media Laboratories Pvt, Ltd., Mumbai, India)].
Five to six colonies of K. pneumoniae suspended in 200 μl double-distilled water and heated at 95°C for 10 min and added with equal proportion of 90% ethanol to DNA to get precipitated. The precipitated top solution was again added with equal proportion of 90% ethanol and centrifuged at 6440 × g for 5 min. Discarded the supernatant and to the deposit again added equal proportion of 90% ethanol and repeated the procedure for two more times. At the end of the procedure, the deposit was left to dry and added double distilled water to remove ethanol and again centrifuged at 6440 × g for 5 min. The deposit with few drops of nuclease-free water was directly used as template DNA for PCR study. The primers used and PCR cycling conditions were described in [Tables
The total concentration of PCR reaction to do single test was 25 μl [PCR ready mix-12.5 μl (Sigma Aldrich, Merck's Life Science), Forward primer-1 μl, Reverse primer-1 μl (Bioserve Hyderabad, India), Template DNA-4 μl and Nuclease free water-6.5 μl (Hi Media Laboratories Pvt, Ltd., Mumbai, India)].
3
Candida albicans Detection Assay
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Grade 1 Whatman (R) filter paper (thickness 180 µm and pore size, 11 µm) was obtained from GE Life Sciences. Scotch BOPP transparent (50.8 mm × 50 meter) tape manufactured by 3 M™, India, was procured from a local stationary shop. Candle sticks were purchased from a local vendor. Neverwet superhydrophobic spray manufactured by Rust-Oleum was purchased from Amazon, India. Glucose (S.L) reagent was purchased from Agappe Diagnostics Ltd. Crystal violet and nuclease-free water were purchased from Himedia. Sodium sulfite (Na2SO3) was purchased from SRL, India. The Candida albicans (ATCC 24433) culture was collected from the Department of Microbiology, Kasturba Medical College, Manipal. Anonymized clinical serum samples (total of 12 samples comprising triplicates of each concentration, specifically, 60 mg/dL, 90 mg/dL, 120 mg/dL, and 150 mg/dL) validated using EM 360 autoanalyzer were collected from the Department of Biochemistry, Kasturba Medical College, Manipal.
4
Antimicrobial Activity and Molecular Identification
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Microorganisms: Alternaria alternata, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Media: Potato Dextrose Agar (PDA, Merck), Bacto agar (Merck), Mueller Hinton Agar (Himedia), Nutrient Agar (Himedia) dan Nutrient Broth (Himedia). Reagents: Deionized water, Tween 20 (Merck), Ethanol (Merck), sodium hypochlorite (Merck), MEthanol (Merck), Streptomycin sulfate (Sigma-Aldrich), PHYTOPure nucleon reagent (Amersham LIFE SCIENCE). Nuclease-Free Water (Himedia), Primer Forward ITS 5 (Macrogen), Primer Reverse ITS 4 (Macrogen), GoTaq Green Master Mix 2X (Promega), Whatman filter paper No. 1 (Sigma-Aldrich), Filter paper for antibacterial test (Oxoid), n-Hexane (Merck), Ethyl acetate (Merck), dimethyl sulfoxide (Sigma-Aldrich), 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich). Instruments: Analytic balance (AND GR-200, GF-2000), Laminar air flow (Sanyo), shaker incubator (Yamato SA320 and IC602), oven (Jisico), Centrifuge (Tomy MX-305), Autoclave (Tomy SX-300), Rotary evaporator (IKA RV10), Microscope (Interscience 4000), Magnetic strirrer (Ayela RCH-3), Varioskan Lux (Thermoscientific), GCMS (Shimadzu-QP 2010 Ultra), PCR (Thermo Scientific Arktik), DNA sequencer (ABI PRISM 3130 Genetic Analyzer).
5
Genomic DNA Extraction Protocol
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Xylene (Product No. 35415; Thermo Fisher Scientific, Waltham, MA, United States); Scalpel/razor blade; ethanol (Cat No. 58051; Jebsen & Jessen GmbH & Co., Hamburg, Germany); Tris HCl (Cat No. 34969; Sisco Research Laboratories Pvt. Ltd., New Delhi, India); EDTA dipotassium salt extrapure (Cat No. 62196; Sisco Research Laboratories Pvt. Ltd.); NaCl (Cat No. 15915; Qualigens Fine Chemicals, Mumbai, India); sodium lauryl sulphate (Cat No. 32096; Sisco Research Laboratories Pvt. Ltd.); proteinase K (Cat No. RM2957; HiMedia Laboratories, LLC, Mumbai, India); phenol molecular biology grade (Cat No. 17286; Sisco Research Laboratories Pvt. Ltd.); chloroform molecular biology grade (Cat No. 96764; Sisco Research Laboratories Pvt. Ltd.); isoamyl alcohol extrapure (Cat No. 69931; Sisco Research Laboratories Pvt. Ltd.); sodium acetate anhydrous extrapure (Cat No. 40104 K05; SDFCL, Bengaluru, India); nuclease-free water (Cat No. ML024; HiMedia Laboratories); mineral oil molecular biology grade (Cat No. MB161; HiMedia Laboratories); agarose low EEO (Cat No. MB002; HiMedia Laboratories); Taq polymerase (Cat No. MBT060A; HiMedia Laboratories); deoxynucleotriphosphates (Cat No. MBT078; HiMedia Laboratories); PCR tubes (Cat No. AB0620; Abgene, Portsmouth, NH, United States); microcentrifuge tubes (Cat No. 509-GRD-Q; QSP by Thermo Fisher Scientific).
6
Optimizing Cellular Assays with Reagent Specifications
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Optiprep density gradient medium, Proteinase K and Phorbol-12-myristate-13-acetate were purchased from Sigma Aldrich (Burlington, MA, USA). THP-1 cell line was procured from NCCS, Pune, India. Pierce BCA Protein Assay Kit, RPMI 1640 media, Fetal Bovine serum and Penicillin–streptomycin, Nunc™ Lab-Tek™ II Chamber Slide™, Lysotracker Green and Prolong Gold Antifade DAPI Mountant, BD cytofix/cytoperm Fixation/Permeabilisation Kit, MaxiSorp Nunc 96 well plates were all from Thermo Fisher Scientific (Waltham, MA, USA).
All oligonucleotides used in the current study were purchased from Integrated DNA Technology (IDT, Coralville, IA, USA). PCR master mix was procured from Takara, Japan. Nuclease free water was obtained from HI Media Laboratories, India. For all buffers/solutions preparation, milliQ water was used. The aptamers population was cloned in a TA cloning vector (pTZ57R/T vector) from Thermo Fisher Scientific, USA, using InsTA Clone PCR cloning Kit. 7H9, 7H11 and OADC were purchased from BD (Franklin Lakes, NJ, USA) and Luria Bertiani media was purchased from HI Media Laboratories, Mumbai, India. Most chemical reagents were purchased from either Sigma Aldrich, USA or HiMedia Laboratories Pvt. Ltd., Mumbai, India.
All oligonucleotides used in the current study were purchased from Integrated DNA Technology (IDT, Coralville, IA, USA). PCR master mix was procured from Takara, Japan. Nuclease free water was obtained from HI Media Laboratories, India. For all buffers/solutions preparation, milliQ water was used. The aptamers population was cloned in a TA cloning vector (pTZ57R/T vector) from Thermo Fisher Scientific, USA, using InsTA Clone PCR cloning Kit. 7H9, 7H11 and OADC were purchased from BD (Franklin Lakes, NJ, USA) and Luria Bertiani media was purchased from HI Media Laboratories, Mumbai, India. Most chemical reagents were purchased from either Sigma Aldrich, USA or HiMedia Laboratories Pvt. Ltd., Mumbai, India.
7
Analyzing Efflux Pumps and Porins in Acinetobacter baumannii
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Real-Time PCR (RT-PCR) was used to analyze the expression of efflux pumps (adeB, adeJ, adeG, adeY, and abeM) and oprD and carO (porins or outer membrane proteins) genes in all the 30 isolates under study. Expression of mRNA was investigated for the efflux pumps and porins genes (Supplementary Table S1 ) using SYBR Green I (Roche, Mannheim, Germany) chemistry according to the manufacturers’ instructions. Each PCR tube (200 µL) had a reaction mixture of following components: 1 pM of each primer; 1 µL of template cDNA (100 ng/µL); 5 µL of Light Cycler 480 SYBR Green I (Roche, Mannheim, Germany) master mix; and 2 µL of nuclease-free water (Hi-Media Laboratories GmbH) in a final volume of 10 µL. PCR was performed for the prepared reactions following the PCR conditions: 1 cycle of 94 °C for 5 min; and 40 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. Analysis for the melt curve was also performed to ensure production of single amplicon after each run. A. baumannii ATCC 19606 and ‘RNA polymerase sigma factor’ gene (rpoD) were used as a reference strain and an internal control gene respectively. All reactions were performed in triplicate and the mean CT value was utilized to analyze the expression level for each isolate.
8
Total RNA Extraction from PBMCs
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The PBMCs obtained by using Histopaque 1077 as well as RBC lysis buffer, and resuspended in 0.5 ml of DEPC -PBS, were used to extract the total cellular RNA using Trizol reagent. 1ml of trizol and 200 µl of chloroform were added to the DEPC -PBS resuspended PBMCs. Mixed well and incubated for 3 min at room temperature.
Centrifuged at 14,000 rpm for 15 min at 4ºC. The upper aqueous phase resulted in the phase seperation was then transferred to a new eppendorf tube and 0.5ml of isopropanol was added, vortexed and placed in -20ºC overnight, followed by centrifugation at 14,000 rpm for 15 min at 4ºC. The obtained pellet was rinsed with ice-cold 70% ethanol and centrifuged at 7,500 rpm for 8 min at 4ºC. The isolated RNA was then reconstituted in nuclease free water (Himedia).
Centrifuged at 14,000 rpm for 15 min at 4ºC. The upper aqueous phase resulted in the phase seperation was then transferred to a new eppendorf tube and 0.5ml of isopropanol was added, vortexed and placed in -20ºC overnight, followed by centrifugation at 14,000 rpm for 15 min at 4ºC. The obtained pellet was rinsed with ice-cold 70% ethanol and centrifuged at 7,500 rpm for 8 min at 4ºC. The isolated RNA was then reconstituted in nuclease free water (Himedia).
9
DNA Nanostructure Synthesis Protocol
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Dulbecco's Modified Eagle Medium (DMEM) was purchased from Lonza. DMEM-F12, HAMS-F12, Fetal bovine serum (FBS), PenStrap, Trypsin-EDTA (0.25%), collagen were purchased from Gibco and Phosphate buffer saline (PBS) was purchased from HyClone. The sequences used for DNA nanostructure synthesis (Table 12345), 6X loading dye, 50 bp DNA ladder, mowiol, transferrin-A488, Cy5, Hoechst, Pitstop-2, Dynasore and Lactose were ordered from Sigma Aldrich. Nuclease free water, ammonium persulfate, ethidium bromide, TEMED, paraformaldehyde and adherent cell culture dishes were purchased from Himedia. Tris-Acetate EDTA (TAE), Acrylamide/bisacrylamide sol 30% were purchased from GeNei. Magnesium chloride was ordered from SRL, India. Galectin-3 was provided as a gift from Johannes team at Institut Curie, Paris.
10
Endothelial Cell Culture and DNA Nanostructures
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1. Materials: Human Umbilical Vein Endothelial Cells (HUVECs), Endothelial Cell Growth Basal Medium-2 (EBM) and EGM-2 SingleQuots TM Supplements were obtained from Lonza. Fetal bovine serum (FBS), PenStrap, Trypsin-EDTA (0.25%), collagen was acquired from Gibco. Phosphate buffer saline (PBS) was purchased from HyClone. The sequences used for DNA nanostructure synthesis (Table 1-4), 6X loading dye, 50 bp DNA ladder, mowiol, transferrin-A488, Hoechst and triton-X 100 were ordered from Sigma Aldrich. Nuclease free water, ammonium persulfate, ethidium bromide, TEMED, paraformaldehyde (37%) and adherent cell culture dishes were procured from Himedia. Tris-Acetate EDTA (TAE), Acrylamide/bisacrylamide sol 30% were purchased from GeNei. Magnesium chloride was ordered from SRL, India. Geltrex TM and CellMask TM were obtained from ThermoFischer. Matrigel and collagen were purchased from Corning.
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